Phospholipases C and D modulate proline accumulation in Thellungiella halophila/salsuginea differently according to the severity of salt or hyperosmotic stress

Proline accumulation is one of the most common responses of plants to environmental constraints. Thellungiella halophila/salsuginea, a model halophyte, accumulates high levels of proline in response to abiotic stress and in the absence of stress. Recently, lipid signaling pathways have been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. Here we investigated the relationship between lipid signaling enzymes and the level of proline in T. salsuginea. Inhibition of phospholipase C (PLC) enzymes by the specific inhibitor U73122 demonstrated that proline accumulation is negatively controlled by PLCs in the absence of stress and under moderate salt stress (200 mM NaCl). The use of 1-butanol to divert some of the phospholipase D (PLD)-derived phosphatidic acid by transphosphatidylation revealed that PLDs exert a positive control on proline accumulation under severe stress (400 mM NaCl or 400 mM mannitol) but have no effect on its accumulation in non-stress conditions. This experimental evidence shows that positive and negative lipid regulatory components are involved in the fine regulation of proline metabolism. These signaling pathways in T. salsuginea are regulated in the opposite sense to those previously described in A. thaliana, revealing that common signaling components affect the physiology of closely related glycophyte and salt-tolerant plants differently.     

Targeted Proteomic Quantification on Quadrupole-Orbitrap Mass Spectrometer

There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and address the pressing demand of systems biology or biomarker evaluation studies.Shotgun proteomics has emerged over the past decade as the most effective method for the qualitative study of complex proteomes (i.e., the identification of the protein content), as illustrated by a wealth of publications (1, 2). In this approach, after enzymatic digestion of the proteins, the generated peptides are analyzed by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)¹ in a data dependent mode. However, the complexity of the digested proteomes under investigation and the wide range of protein abundances limit the reproducibility and the sensitivity of this stochastic approach (3), which is critical if one aims at the systematic quantification of the proteins. Thus, alternative MS approaches have emerged for the systematic quantitative study of complex proteomes, the MS-based targeted proteomics (4). In this hypothesis-driven approach, only specific subsets of analytes (a few targeted peptides used as surrogates for the proteins of interest) are selectively measured in predefined m/z ranges and retention time windows, which overcomes the bias toward most abundant compounds commonly observed with shotgun proteomics. When applied to complex biological samples—for example, bodily fluids such as urine or plasma—targeted proteomics requires high performance instruments allowing measurements of a wide dynamic range (many orders of magnitude), with high sensitivity in order to detect peptides in the low amol range and sufficient selectivity to cope with massive biochemical background (5). Selected reaction monitoring (SRM) on triple quadrupole (6) or triple quadrupole-linear ion trap mass spectrometers (7) has emerged as a means to conduct such analyses (8). Initially applied in the MS analysis of small molecules (9, 10), SRM has gradually emerged as the reference quantitative technique for analyzing proteins (or peptides) in biological samples. When coupled with the isotope dilution strategy (11, 12), this very effective technique allows the precise quantification of proteins (13–18). However, despite the increased selectivity provided by the two-stage mass filtering of SRM (at the precursor and fragment ion levels), the low resolution of mass selection does not allow the systematic removal of interferences (19, 20). Moreover, in proteomics, the biochemical background has a composition similar to that of the analytes of interest, which remains a major hurdle limiting the sensitivity of assays, especially in a bodily fluid matrix. High resolution/accurate mass (HR/AM) analysis represents a promising alternative approach that might more efficiently distinguish the compounds of interest from interferences in targeted proteomics. Such analyses can be conducted on orbitrap-based mass spectrometers because of their high sensitivity and high mass accuracy capabilities (21). The introduction of the benchtop standalone orbitrap mass spectrometer (Exactive) (22) further strengthened the attractiveness of the approach, especially in the field of small molecule analysis (23, 24). However, as quantification using trapping devices intrinsically suffers from a limited dynamic range because of the overall ion capacity, the complexity of biological samples remains very challenging even with the HR/AM approach (25). Targeted protein analysis with triple quadrupole mass spectrometers keeps on showing significant superiority for such samples.² The recently developed quadrupole-orbitrap mass spectrometer (Q-Exactive) can potentially address this issue.³ It is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection (26, 27). This configuration combines advantages of triple quadrupole instruments for mass filtering and orbitrap-based mass spectrometers for HR/AM measurement. The ability of the instrument to select a restricted m/z range or (sequentially) a small number of precursor ions offers new opportunities for quantification in complex samples by selectively enriching low abundant components. The resulting data, acquired in the so-called single ion monitoring (SIM) mode, fully benefit from the trapping capability while keeping a high acquisition rate as a result of the fast switching time between targeted precursor ions of the quadrupole. Although this mode of data acquisition is possible with a configuration combining a linear ion trap with the orbitrap (as in the LTQ-Orbitrap mass spectrometer), its effectiveness is far more limited in this case. The quadrupole-orbitrap configuration presents significant benefits by selectively isolating a narrow population of precursor ions. Other features of the instrument include its multiplexed trapping capability (26) using either the C-trap or the higher energy collisional dissociation (HCD) cell (28, 29), which opens new avenues in the design of innovative acquisition methods for quantification studies. For the first time, a panel of acquisition methods is designed and applied to targeted quantification at the MS and MS/MS levels. In the latter case, the simultaneous monitoring of multiple MS/MS fragmentation channels, also called parallel reaction monitoring⁴ (PRM), is particularly promising for quantifying large sets of peptides with increased selectivity.     

Life history traits variation in heterogeneous environment: The case of a freshwater snail resistance to pond drying

Ecologists and population geneticists have long suspected that the diversity of living organisms was connected to the structure of their environment. In heterogeneous environments, diversifying selection combined to restricted gene flow may indeed lead to locally adapted populations. The freshwater snail, Galba truncatula, is a good model to address this question because it is present in a heterogeneous environment composed of temporary and permanent waters. In order to test the selective importance of those environments, we proposed here to measure survival of lineages from both habitats during drought episodes. To this purpose, we experimentally submitted adults and juveniles individuals from both habitats to drought. We found a difference in desiccation resistance between temporary and permanents waters only for adults. Adults from temporary habitats were found more resistant to drought. This divergence in desiccation resistance seems to explain the unexpected life history traits differences between habitats observed.     

A theory of germinal center B cell selection, division, and exit

High-affinity antibodies are generated in germinal centers in a process involving mutation and selection of B cells. Information processing in germinal center reactions has been investigated in a number of recent experiments. These have revealed cell migration patterns, asymmetric cell divisions, and cell-cell interaction characteristics, used here to develop a theory of germinal center B cell selection, division, and exit (the LEDA model). According to this model, B cells selected by T follicular helper cells on the basis of successful antigen processing always return to the dark zone for asymmetric division, and acquired antigen is inherited by one daughter cell only. Antigen-retaining B cells differentiate to plasma cells and leave the germinal center through the dark zone. This theory has implications for the functioning of germinal centers because compared to previous models, high-affinity antibodies appear one day earlier and the amount of derived plasma cells is considerably larger.     

MCM8- and MCM9-Deficient Mice Reveal Gametogenesis Defects and Genome Instability Due to Impaired Homologous Recombination

We generated knockout mice for MCM8 and MCM9 and show that deficiency for these genes impairs homologous recombination (HR)-mediated DNA repair during gametogenesis and somatic cells cycles. MCM8(-/-) mice are sterile because spermatocytes are blocked in meiotic prophase I, and females have only arrested primary follicles and frequently develop ovarian tumors. MCM9(-/-) females also are sterile as ovaries are completely devoid of oocytes. In contrast, MCM9(-/-) testes produce spermatozoa, albeit in much reduced quantity. Mcm8(-/-) and Mcm9(-/-) embryonic fibroblasts show growth defects and chromosomal damage and cannot overcome a transient inhibition of replication fork progression. In these cells, chromatin recruitment of HR factors like Rad51 and RPA is impaired and HR strongly reduced. We further demonstrate that MCM8 and MCM9 form a complex and that they coregulate their stability. Our work uncovers essential functions of MCM8 and MCM9 in HR-mediated DSB repair during gametogenesis, replication fork maintenance, and DNA repair.     

Engineering of three-finger fold toxins creates ligands with original pharmacological profiles for muscarinic and adrenergic receptors

Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test "loop grafting," a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α(1A)-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α(1A)-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles.