The high-resolution NMR structure of the R21A Spc-SH3:P41 complex: Understanding the determinants of binding affinity by comparison with Abl-SH3

Background  

SH3 domains are small protein modules of 60–85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lower affinity for the α-spectrin SH3 domain (Spc-SH3).     

Yif1B Is Involved in the Anterograde Traffic Pathway and the Golgi Architecture

Yif1B is an intracellular membrane‐bound protein belonging to the Yip family, shown previously to control serotonin 5‐HT_1A receptor targeting to dendrites. Because some Yip proteins are involved in the intracellular traffic between the ER and the Golgi, here we investigated the precise localization of Yif1B in HeLa cells. We found that Yif1B is not resident into the Golgi, but rather belongs to the IC compartment. After analyzing the role of Yif1B in protein transport, we showed that the traffic of the VSVG protein marker was accelerated in Yif1B depleted HeLa cells, as well as in hippocampal neurons from Yif1B KO mice. Conversely, Yif1B depletion in HeLa cells did not change the retrograde traffic of ShTx. Interestingly, in long term depletion of Yif1B as in Yif1B KO mice, we observed a disorganized Golgi architecture in CA1 pyramidal hippocampal neurons, which was confirmed by electron microscopy. However, because short term depletion of Yif1B did not change Golgi structure, it is likely that the implication of Yif1B in anterograde traffic does not rely on its role in structural organization of the Golgi apparatus, but rather on its shuttling between the ER, the IC and the Golgi compartments.      

Insulin resistance induced by hydrocortisone is increased in patients with abdominal obesity

Glucocorticoids hypersensitivity may be involved in the development of abdominal obesity and insulin resistance. Eight normal weight and eight obese women received on two occasions a 3-h intravenous infusion of saline or hydrocortisone (HC) (1.5 microg x kg(-1) x min(-1)). Plasma cortisol, insulin, and glucose levels were measured every 30 min from time(-30) (min) (time(-30)) to time(240). Free fatty acids, adiponectin, and plasminogen activator inhibitor-1 (PAI-1) levels were measured at time(-30), time(180), and time(240). At time(240), subjects underwent an insulin tolerance test to obtain an index of insulin sensitivity (K(ITT)). Mean(30-240) cortisol level was similar in control and obese women after saline (74 +/- 16 vs. 75 +/- 20 microg/l) and HC (235 +/- 17 vs. 245 +/- 47 microg/l). The effect of HC on mean(180-240) insulin, mean(180-240) insulin resistance obtained by homeostasis model assessment (HOMA-IR), and K(ITT) was significant in obese (11.4 +/- 2.0 vs. 8.2 +/- 1.3 mU/l, P < 0.05; 2.37 +/- 0.5 vs. 1.64 +/- 0.3, P < 0.05; 2.81 +/- 0.9 vs. 3.32 +/- 1.02%/min, P < 0.05) but not in control women (3.9 +/- 0.6 vs. 2.8 +/- 0.5 mU/l; 0.78 +/- 0.1 vs. 0.49 +/- 0.1; 4.36 +/- 1.1 vs. 4.37 +/- 1.2%/min). In the whole population, the quantity of visceral fat, estimated by computerized tomography scan, was correlated with the increment of plasma insulin and HOMA-IR during HC infusion [Delta mean(30-240) insulin (r = 0.61, P < 0.05), Delta mean(30-240) HOMA-IR (r = 0.66, P < 0.01)]. The increase of PAI-1 between time(180) and time(240) after HC was higher in obese women (+25%) than in controls (+12%) (P < 0.05), whereas no differential effect between groups was observed for free fatty acids or adiponectin. A moderate hypercortisolism, equivalent to that induced by a mild stress, has more pronounced consequences on insulin sensitivity in abdominally obese women than in controls. These deleterious effects are correlated with the amount of visceral fat.     

Species delimitation of the liquorice tribe (Leguminosae: Glycyrrhizeae) based on phylogenomic and machine learning analyses

The liquorice tribe Glycyrrhizeae is a leguminous herbaceous group of plants comprised of the genera Glycyrrhiza and Glycyrrhizopsis. Some Glycyrrhiza taxa contain glycyrrhizin, a pharmacologically significant sweet substance that also has applications in crafting industrial materials. Here, we utilized an expanded taxon sampling of Glycyrrhizeae to reconstruct the phylogenetic relationships in the tribe based on genome skimming data, including whole chloroplast genomes, nuclear ribosomal DNA, and low-copy nuclear DNA. We also launched machine learning analysis (MLA) for one species pair with controversial taxonomic boundary. The integrated results indicated Glycyrrhizopsis should be split from Glycyrrhiza, while the former genus Meristotropis should be treated as part of Glycyrrhiza. Glycyrrhizopsis includes two species, Glycyrrhizopsis asymmetrica and Glycyrrhizopsis flavescens, and we recognize 13 species in Glycyrrhiza: Glycyrrhiza acanthocarpa, Glycyrrhiza astragalina, Glycyrrhiza bucharica, Glycyrrhiza echinata, Glycyrrhiza foetida, Glycyrrhiza glabra, Glycyrrhiza gontscharovii, Glycyrrhiza lepidota, Glycyrrhiza macedonica, Glycyrrhiza pallidiflora, Glycyrrhiza squamulosa, Glycyrrhiza triphylla, and Glycyrrhiza yunnanensis. We propose a broader G. glabra that includes former Glycyrrhiza aspera, G. glabra s.s., Glycyrrhiza inflata, and Glycyrrhiza uralensis, and represents the glycyrrhizin-contained medicinal group. Our ancestral state inferences show the ancestor of Glycyrrhiza lacked glycyrrhizin, and the presence of glycyrrhizin evolved twice within Glycyrrhiza during the last one million years. Our integrative phylogenomics-MLA study not only provides new insights into long-standing taxonomic controversies of Glycyrrhizeae, but also represents a useful approach for future taxonomic studies on other plant taxa.     

Rearrangements of integral membrane components during in vitro aging of sheep erythrocyte membranes

In vitro aged sheep erythrocytes and sheep erythrocyte ghosts spontaneously release vesicles that consist of long protrusions affixed to flattened headlike structures. The intramembranous particles seen on the protoplasmic face of freeze fracture electron micrographs of vesicle protrusions are arranged in paired particle rows. On the equivalent fracture face of headlike structures, the particle density is low; if particles are present, they are clustered along the rim of the flattened headlike structure and at the junction with the protrusion. The released vesicles are depleted of the intramembranous particles seen on the exoplasmic face of ghost but retain almost exclusively particles of the protoplasmic face. Correspondingly, the exoplasmic face of ghosts that have released vesicles reveals a 28 percent higher density of intramembranous particles than that of fresh ghosts. Purified vesicles are depleted of spectrin but retain integral membrane proteins, with one of an apparent mol wt of 160,000 accounting for nearly 50 percent of the total protein (Lutz, H.U.,R. Barber, and R.F. McGuire. 1976. J. Biol. Chem. 251:3500-3510). When vesicles are modified with the cleavable cross-linking reagent [(35)S]dithiobis (succinimidyl propionate)at 0 degrees C, the 160,000 mol wt protein is rapidly converted to disulfide-linked dimers and higher oligomers. Exposure of intact ghosts to the reagent in the same way fails to yield equivalent polymers. A comparison of the morphological and biochemical aspects of ghosts and vesicles suggest that a marked rearrangement of membrane proteins accompanies the supramolecular redistribution of intramembranous particles during spontaneous vesiculation. The results also suggest that the paired particles of the protoplasmic face of vesicle protrusions are arranged in paired helices and contain the 160,000 mol wt protein as dimers.     

Phenotypic suppression of morphogenetic mutants of Dictyostelium discoideum.

The effects of cAMP pulses on the capacity of 15 aggregateless mutants to differentiate and construct fruiting bodies are compared to those obtained when mutant cells are starved with wild-type amoebae. Mutant strains are classified into three main groups depending upon the degree to which their phenotypic defects can be corrected. These data extend studies published earlier [Darmon, M., Brachet, P., and Pereira da Silva, L. (1975). Chemotactic signals induce cell differentiation in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA72, 3163–3166; Pereira da Silva, L., Darmon, M., Brachet, P., Klein, C., and Barrand, P. (1975). Induction of cell differentiation by the chemotactic signal in Dictyostelium discoideum. In “Proceedings of the Tenth FEBS Meeting,” pp. 269–276]. (1) Only one mutant was unresponsive both to cAMP pulses and to the presence of wild-type amoebae and did not display any of the properties of differentiated cells. (2) Following treatment with cAMP pulses, 11 mutants developed certain properties of aggregation-competent amoebae. They increased their levels of cellular phosphodiesterase, showed an enhanced chemotactic sensitivity to cAMP, and established specific cell contacts. None of these amoebae could differentiate further. They did co-aggregate to some extent with wild-type cells, but failed to differentiate into spores. Rather, mutant cells were excluded from the pseudoplasmodium during the process of morphogenesis of the fruiting body. (3) In contrast, the aggregateless phenotype of three mutants was fully corrected by both cAMP pulses and the presence of wild-type cells. These findings are discussed on the basis of a relationship between the chemotactic signal and cell differentiation.     

Binding of Concanavalin A and its effect on the differentiation of Dictyostelium Discoideum.

Dictyostelium discoideum amoebae have two classes of binding sites for concanavalin A, one accessible to a competitive inhibitor of concanavalin A binding and one inaccessible. Uptake of concanavalin A occurs and is associated with a transfer from accessible to inaccessible binding sites. Low amounts of concanavalin A bound to cells produce a slight stimulation of differentiation, and this is associated with an increase in production of phosphodiesterase. Above a critical amount of concanavalin A bound, cell differentiation and phosphodiesterase production are inhibited. The relationship of concanavalin A-induced membrane changes to cell differentation is discussed.     

Control of GT repeat stability in Schizosaccharomyces pombe by mismatch repair factors

The mismatch repair (MMR) system ensures genome integrity by removing mispaired and unpaired bases that originate during replication. A major source of mutational changes is strand slippage in repetitive DNA sequences without concomitant repair. We established a genetic assay that allows measuring the stability of GT repeats in the ade6 gene of Schizosaccharomyces pombe. In repair-proficient strains most of the repeat variations were insertions, with addition of two nucleotides being the most frequent event. GT repeats were highly destabilized in strains defective in msh2 or pms1. In these backgrounds, mainly 2-bp insertions and 2-bp deletions occurred. Surprisingly, essentially the same high mutation rate was found with mutants defective in msh6. In contrast, a defect in swi4 (a homologue of Msh3) caused only slight effects, and instability was not further increased in msh6 swi4 double mutants. Also inactivation of exo1, which encodes an exonuclease that has an MMR-dependent function in repair of base-base mismatches, caused only slightly increased repeat instability. We conclude that Msh2, Msh6, and Pms1 have an important role in preventing tract length variations in dinucleotide repeats. Exo1 and Swi4 have a minor function, which is at least partially independent of MMR.     

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.