Second sialic acid binding site in Newcastle disease virus hemagglutinin-neuraminidase: implications for fusion

Paramyxoviruses are the leading cause of respiratory disease in children. Several paramyxoviruses possess a surface glycoprotein, the hemagglutinin-neuraminidase (HN), that is involved in attachment to sialic acid receptors, promotion of fusion, and removal of sialic acid from infected cells and progeny virions. Previously we showed that Newcastle disease virus (NDV) HN contained a pliable sialic acid recognition site that could take two states, a binding state and a catalytic state. Here we present evidence for a second sialic acid binding site at the dimer interface of HN and present a model for its involvement in cell fusion. Three different crystal forms of NDV HN now reveal identical tetrameric arrangements of HN monomers, perhaps indicative of the tetramer association found on the viral surface.     

Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase

Paramyxoviruses are the main cause of respiratory disease in children. One of two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN), has several functions in addition to being the major surface antigen that induces neutralizing antibodies. Here we present the crystal structures of Newcastle disease virus HN alone and in complex with either an inhibitor or with the beta-anomer of sialic acid. The inhibitor complex reveals a typical neuraminidase active site within a beta-propeller fold. Comparison of the structures of the two complexes reveal differences in the active site, suggesting that the catalytic site is activated by a conformational switch. This site may provide both sialic acid binding and hydrolysis functions since there is no evidence for a second sialic acid binding site in HN. Evidence for a single site with dual functions is examined and supported by mutagenesis studies. The structure provides the basis for the structure-based design of inhibitors for a range of paramyxovirus-induced diseases.     

Influence of decorin and biglycan on mechanical properties of multiple tendons in knockout mice

Evaluations of tendon mechanical behavior based on biochemical and structural arrangement have implications for designing tendon specific treatment modalities or replacement strategies. In addition to the well studied type I collagen, other important constituents of tendon are the small proteoglycans (PGs). PGs have been shown to vary in concentration within differently loaded areas of tendon, implicating them in specific tendon function. This study measured the mechanical properties of multiple tendon tissues from normal mice and from mice with knock-outs of the PGs decorin or biglycan. Tail tendon fascicles, patellar tendons (PT), and flexor digitorum longus tendons (FDL), three tissues representing different in vivo loading environments, were characterized from the three groups of mice. It was hypothesized that the absence of decorin or biglycan would have individual effects on each type of tendon tissue. Surprisingly, no change in mechanical properties was observed for the tail tendon fascicles due to the PG knockouts. The loss of decorin affected the PT causing an increase in modulus and stress relaxation, but had little effect on the FDL. Conversely, the loss of biglycan did not significantly affect the PT, but caused a reduction in both the maximum stress and modulus of the FDL. These results give mechanical support to previous biochemical data that tendons likely are uniquely tailored to their specific location and function. Variances such as those presented here need to be further characterized and taken into account when designing therapies or replacements for any one particular tendon.     

Inherent dynamics within the Crimean-Congo Hemorrhagic fever virus protease are localized to the same region as substrate interactions

Crimean-Congo Hemorrhagic fever virus (CCHFV) is one of several lethal viruses that encodes for a viral ovarian tumor domain (vOTU), which serves to cleave and remove ubiquitin (Ub) and interferon stimulated gene product 15 (ISG15) from numerous proteins involved in cellular signaling. Such manipulation of the host cell machinery serves to downregulate the host response and, therefore, complete characterization of these proteases is important. While several structures of the CCHFV vOTU protease have been solved, both free and bound to Ub and ISG15, few structural differences have been found and little insight has been gained as to the structural plasticity of this protease. Therefore, we have used NMR relaxation experiments to probe the dynamics of CCHFV vOTU, both alone and in complex with Ub, discovering a highly dynamic protease that exhibits conformational exchange within the same regions found to engage its Ub substrate. These experiments reveal a structural plasticity around the N-terminal regions of CCHFV vOTU, which are unique to vOTUs, and provide a rationale for engaging multiple substrates with the same binding site.     

Changes in Socioeconomic Inequality in Indonesian Children’s Cognitive Function from 2000 to 2007: A Decomposition Analysis

Background

Measuring social inequalities in health is common; however, research examining inequalities in child cognitive function is more limited. We investigated household expenditure-related inequality in children’s cognitive function in Indonesia in 2000 and 2007, the contributors to inequality in both time periods, and changes in the contributors to cognitive function inequalities between the periods.

Methods

Data from the 2000 and 2007 round of the Indonesian Family Life Survey (IFLS) were used. Study participants were children aged 7–14 years (n = 6179 and n = 6680 in 2000 and 2007, respectively). The relative concentration index (RCI) was used to measure the magnitude of inequality. Contribution of various contributors to inequality was estimated by decomposing the concentration index in 2000 and 2007. Oaxaca-type decomposition was used to estimate changes in contributors to inequality between 2000 and 2007.

Results

Expenditure inequality decreased by 45% from an RCI = 0.29 (95% CI 0.22 to 0.36) in 2000 to 0.16 (95% CI 0.13 to 0.20) in 2007 but the burden of poorer cognitive function was higher among the disadvantaged in both years. The largest contributors to inequality in child cognitive function were inequalities in per capita expenditure, use of improved sanitation and maternal high school attendance. Changes in maternal high school participation (27%), use of improved sanitation (25%) and per capita expenditures (18%) were largely responsible for the decreasing inequality in children’s cognitive function between 2000 and 2007.

Conclusions

Government policy to increase basic education coverage for women along with economic growth may have influenced gains in children’s cognitive function and reductions in inequalities in Indonesia.     

Structure and dynamics of pin1 during catalysis by NMR

The link between internal enzyme motions and catalysis is poorly understood. Correlated motions in the microsecond-to-millisecond timescale may be critical for enzyme function. We have characterized the backbone dynamics of the peptidylprolyl isomerase (Pin1) catalytic domain in the free state and during catalysis. Pin1 is a prolyl isomerase of the parvulin family and specifically catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds. Pin1 has been shown to be essential for cell-cycle progression and to interact with the neuronal tau protein inhibiting its aggregation into fibrillar tangles as found in Alzheimer's disease. (15)N relaxation dispersion measurements performed on Pin1 during catalysis reveal conformational exchange processes in the microsecond timescale. A subset of active site residues undergo kinetically similar exchange processes even in the absence of a substrate, suggesting that this area is already "primed" for catalysis. Furthermore, structural data of the turning-over enzyme were obtained through inter- and intramolecular nuclear Overhauser enhancements. This analysis together with a characterization of the substrate concentration dependence of the conformational exchange allowed the distinguishing of regions of the enzyme active site that are affected primarily by substrate binding versus substrate isomerization. Together these data suggest a model for the reaction trajectory of Pin1 catalysis.     

Multilocus evidence for globally distributed cryptic species and distinct populations across ocean gyres in a mesopelagic copepod

Zooplanktonic taxa have a greater number of distinct populations and species than might be predicted based on their large population sizes and open‐ocean habitat, which lacks obvious physical barriers to dispersal and gene flow. To gain insight into the evolutionary mechanisms driving genetic diversification in zooplankton, we developed eight microsatellite markers to examine the population structure of an abundant, globally distributed mesopelagic copepod, Haloptilus longicornis, at 18 sample sites across the Atlantic and Pacific Oceans (n = 761). When comparing our microsatellite results with those of a prior study that used a mtDNA marker (mtCOII, n = 1059, 43 sample sites), we unexpectedly found evidence for the presence of a cryptic species pair. These species were globally distributed and apparently sympatric, and were separated by relatively weak genetic divergence (reciprocally monophyletic mtCOII lineages 1.6% divergent; microsatellite F_ST ranging from 0.28 to 0.88 across loci, P < 0.00001). Using both mtDNA and microsatellite data for the most common of the two species (n = 669 for microsatellites, n = 572 for mtDNA), we also found evidence for allopatric barriers to gene flow within species, with distinct populations separated by continental landmasses and equatorial waters in both the Atlantic and Pacific Ocean basins. Our study shows that oceanic barriers to gene flow can act as a mechanism promoting allopatric diversification in holoplanktonic taxa, despite the high potential dispersal abilities and pelagic habitat for these species.     

Macroautophagy Abnormality in Essential Tremor

Macroautophagy is a cellular mechanism for the clearance of protein aggregates and damaged organelles. Impaired macroautophagy has been observed in neurodegenerative disorders. We investigated the macroautophagy pathway in essential tremor (ET) cases compared to age-matched controls. We analyzed microtubule-associated protein light chain 3-II (LC3-II), S6K, phosphorylated S6K, beclin-1, and mitochondrial membrane proteins levels by Western blot in the post-mortem cerebellum of 10 ET cases and 11 controls. We also performed immunohistochemistry in 12 ET cases and 13 controls to quantify LC3 clustering in Purkinje cells (PCs). LC3-II protein levels were significantly lower in ET cases vs. controls on Western blot (0.84±0.14 vs. 1.00±0.14, p = 0.02), and LC3-II clustering in PCs by immunohistochemistry was significantly lower in ET cases vs. controls (2.03±3.45 vs. 8.80±9.81, p = 0.03). In ET cases, disease duration was inversely correlated with LC3-II protein level (r = −0.64, p = 0.046). We found that mitochondrial membrane proteins were accumulated in ET (TIM23: 1.36±0.11 in ET cases vs. 1.00±0.08 in controls, p = 0.02; TOMM20: 1.63±0.87 in ET cases vs. 1.00±0.14 in controls, p = 0.03). Beclin-1, which is involved in macroautophagy, was strikingly deficient in ET (0.42±0.13 vs. 1.00±0.35, p<0.001). Decreased macroautophagy was observed in the ET cerebellum, and this could be due to a decrease in beclin-1 levels, which subsequently lead to mitochondrial accumulation as a result of autophagic failure. This provides a possible means by which perturbed macroautophagy could contribute to PC pathology in ET.