Oligosaccharide and sucrose complexes of amylosucrase. Structural implications for the polymerase activity

The glucosyltransferase amylosucrase is structurally quite similar to the hydrolase alpha-amylase. How this switch in functionality is achieved is an important and fundamental question. The inactive E328Q amylosucrase variant has been co-crystallized with maltoheptaose, and the structure was determined by x-ray crystallography to 2.2 A resolution, revealing a maltoheptaose binding site in the B'-domain somewhat distant from the active site. Additional soaking of these crystals with maltoheptaose resulted in replacement of Tris in the active site with maltoheptaose, allowing the mapping of the -1 to +5 binding subsites. Crystals of amylosucrase were soaked with sucrose at different concentrations. The structures at approximately 2.1 A resolution revealed three new binding sites of different affinity. The highest affinity binding site is close to the active site but is not in the previously identified substrate access channel. Allosteric regulation seems necessary to facilitate access from this binding site. The structures show the pivotal role of the B'-domain in the transferase reaction. Based on these observations, an extension of the hydrolase reaction mechanism valid for this enzyme can be proposed. In this mechanism, the glycogen-like polymer is bound in the widest access channel to the active site. The polymer binding introduces structural changes that allow sucrose to migrate from its binding site into the active site and displace the polymer.     

A simplified method for the detection of Y chromosome microdeletions in infertile men using a multiplex sequence-tagged site-based amplification

Sixteen sequence-tagged sites (STSs) were combined in five amplification reactions, to screen for deletions of DNA fragments located within the AZFa, AZFb, and AZFc regions of the Y chromosome. This multiplex strategy is fast and reliable, and most of the azoospermia-associated deletions reported so far are detected with this simplified method. Internal control STSs are included that allow discrimination between deletion and failure of amplification.     

Improved proteomic discovery by sample pre-fractionation using dual-column ion-exchange high performance liquid chromatography

Clinically relevant biomarkers are urgently needed for improving patient diagnosis, risk stratification, prognosis and therapeutic treatments. There is a particularly compelling motivation for identifying protein-based indicators of early-stage disease for more effective interventions. Despite recent progress, the proteomic discovery process remains a daunting challenge due to the sheer heterogeneity and skewed protein abundances in biofluids. Even the most advanced mass spectrometry systems exhibit limiting overall dynamic ranges and sensitivities relative to the needs of modern biomedical applications. To this end, we report the development of a robust, rapid, and reproducible high performance ion-exchange liquid chromatography pre-fractionation method that allows for improved proteomic detection coverage of complex biological specimens using basic tandem mass spectrometry screening procedures. This form of sample simplification prior to global proteomic profiling, which we refer to collectively as 'fractionomics', increases the number and diversity of proteins that can be confidently identified in tissue and cell lysates as compared to the straight analysis of unfractionated crude extracts.     

Selective blockade of NF-kappa B activity in airway immune cells inhibits the effector phase of experimental asthma

Knockout mice studies have revealed that NF-kappaB plays a critical role in Th2 cell differentiation and is therefore required for induction of allergic airway inflammation. However, the questions of whether NF-kappaB also plays a role in the effector phase of airway allergy and whether inhibiting NF-kappaB could have therapeutic value in the treatment of established asthma remain unanswered. To address these issues, we have assessed in OVA-sensitized wild-type mice the effects of selectively antagonizing NF-kappaB activity in the lungs during OVA challenge. Intratracheal administration of NF-kappaB decoy oligodeoxynucleotides to OVA-sensitized mice led to efficient nuclear transfection of airway immune cells, but not constitutive lung cells and draining lymph node cells, associated with abrogation of NF-kappaB activity in the airways upon OVA provocation. NF-kappaB inhibition was associated with strong attenuation of allergic lung inflammation, airway hyperresponsiveness, and local production of mucus, IL-5, IL-13, and eotaxin. IL-4 and OVA-specific IgE and IgG1 production was not reduced. This study demonstrates for the first time that activation of NF-kappaB in local immune cells is critically involved in the effector phase of allergic airway disease and that specific NF-kappaB inhibition in the lungs has therapeutic potential in the control of pulmonary allergy.     

PIK3R1 Mutations Cause Syndromic Insulin Resistance with Lipoatrophy

Short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome is a developmental disorder with an unknown genetic cause and hallmarks that include insulin resistance and lack of subcutaneous fat. We ascertained two unrelated individuals with SHORT syndrome, hypothesized that the observed phenotype was most likely due to de novo mutations in the same gene, and performed whole-exome sequencing in the two probands and their unaffected parents. We then confirmed our initial observations in four other subjects with SHORT syndrome from three families, as well as 14 unrelated subjects presenting with syndromic insulin resistance and/or generalized lipoatrophy associated with dysmorphic features and growth retardation. Overall, we identified in nine affected individuals from eight families de novo or inherited PIK3R1 mutations, including a mutational hotspot (c.1945C>T [p.Arg649Trp]) present in four families. PIK3R1 encodes the p85α, p55α, and p50α regulatory subunits of class IA phosphatidylinositol 3 kinases (PI3Ks), which are known to play a key role in insulin signaling. Functional data from fibroblasts derived from individuals with PIK3R1 mutations showed severe insulin resistance for both proximal and distal PI3K-dependent signaling. Our findings extend the genetic causes of severe insulin-resistance syndromes and provide important information with respect to the function of PIK3R1 in normal development and its role in human diseases, including growth delay, Rieger anomaly and other ocular affections, insulin resistance, diabetes, paucity of fat, and ovarian cysts.     

Probing the different chaperone activities of the bacterial HSP70-HSP40 system using a thermolabile luciferase substrate

During mild heat-stress, a native thermolabile polypeptide may partially unfold and transiently expose water-avoiding hydrophobic segments that readily tend to associate into a stable misfolded species, rich in intra-molecular non-native beta-sheet structures. When the concentration of the heat-unfolded intermediates is elevated, the exposed hydrophobic segments tend to associate with other molecules into large stable insoluble complexes, also called "aggregates." In mammalian cells, stress- and mutation-induced protein misfolding and aggregation may cause degenerative diseases and aging. Young cells, however, effectively counteract toxic protein misfolding with a potent network of molecular chaperones that bind hydrophobic surfaces and actively unfold otherwise stable misfolded and aggregated polypeptides. Here, we followed the behavior of a purified, initially mostly native thermolabile luciferase mutant, in the presence or absence of the Escherichia coli DnaK-DnaJ-GrpE chaperones and/or of ATP, at 22 °C or under mild heat-stress. We concomitantly measured luciferase enzymatic activity, Thioflavin-T fluorescence, and light-scattering to assess the effects of temperature and chaperones on the formation, respectively, of native, unfolded, misfolded, and/or of aggregated species. During mild heat-denaturation, DnaK-DnaJ-GrpE+ATP best maintained, although transiently, high luciferase activity and best prevented heat-induced misfolding and aggregation. In contrast, the ATP-less DnaK and DnaJ did not maintain optimal luciferase activity and were less effective at preventing luciferase misfolding and aggregation. We present a model accounting for the experimental data, where native, unfolded, misfolded, and aggregated species spontaneously inter-convert, and in which DnaK-DnaJ-GrpE+ATP specifically convert stable misfolded species into unstable unfolded intermediates.     

Semaphorin 3F and Neuropilin-2 Control the Migration of Human T-Cell Precursors

Neuropilins and semaphorins are known as modulators of axon guidance, angiogenesis, and organogenesis in the developing nervous system, but have been recently evidenced as also playing a role in the immune system. Here we describe the expression and role of semaphorin 3F (SEMA3F) and its receptor neuropilin-2 (NRP2) in human T cell precursors. NRP2 and SEMA3F are expressed in the human thymus, in both lymphoid and non-lymphoid compartments. SEMA3F have a repulsive effect on thymocyte migration and inhibited CXCL12- and sphingosine-1-phosphate (S1P)-induced thymocyte migration by inhibiting cytoskeleton reorganization prior to stimuli. Moreover, NRP2 and SEMA3F are expressed in human T-cell acute lymphoblastic leukemia/lymphoma primary cells. In these tumor cells, SEMA3F also blocks their migration induced by CXCL12 and S1P. Our data show that SEMA3F and NRP2 are further regulators of human thymocyte migration in physiological and pathological conditions.     

What is the nature of the replicative niche of a stealthy bug named Brucella?

Brucella spp. are facultatively intracellular bacteria that persist and multiply in the macrophages of their mammalian hosts. The so-called phagosome to which they have adapted is their natural living niche. Characterization of this niche would facilitate an understanding of the true relationship between the host cell and the intracellular bacteria. This Opinion analyses and discusses the characteristic properties and genesis of this vacuole during phagocytosis as deduced from the virulence factors necessary for intracellular multiplication of the pathogen. We conclude that the replicative niche of Brucella spp.--the 'brucellosome'--differs from all other cellular organelles, and that it isolates the pathogen from certain cytoplasmic nutrients. Adaptation to the stress conditions encountered and the use of anaerobic respiration enable brucellae to replicate in the compartment they create.     

Preparing to Work with Big Data in Proteomics – A Report on the HUPO‐PSI Spring Workshop

The Human Proteome Organisation Proteomics Standards Initiative (HUPO‐PSI) was established in 2002 with the aim of defining community standards for data representation in proteomics and facilitating data comparison, exchange and verification. The 2013 annual spring workshop was hosted by the University of Liverpool, UK and concentrated on updating and refining the existing standards in the light of new methodologies and technologies. To control the inflation of file sizes, strategies for file compression, particularly for mzML files, were explored. Best practices for encoding information such as protein grouping and PTM localisation were refined and documented. Additional example files for the mzQuantML format were designed to provide support for selected reaction monitoring techniques. Enhancements to the PSI Common Query Interface (PSICQUIC) and PSI‐MI XML were discussed. Finally, the group engaged in discussion on how the existing work of the HUPO‐PSI can be leveraged by the Metabolomics Standards Initiative to improve the capture of metabolite data.