Identification and SAR of squarate inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK-2)

A novel series of inhibitors for mitogen activated protein kinase-activated protein kinase 2 (MK-2) are reported. These squarate based inhibitors were identified via a high-throughput screen. An MK2 co-structure with the starting ligand was obtained and a structure based approach was followed to optimize potency and selectivity.     

7A projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals

Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7A resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.     

Atomic-level characterization of disordered protein ensembles

The roles of unfolded states of proteins in normal folding and in diseases involving aggregation, as well as the prevalence and regulatory functions of intrinsically disordered proteins, have become increasingly recognized. The structural representation of these disordered states as ensembles of interconverting conformers can therefore provide critical insights. Experimental methods can be used to probe ensemble-averaged structural properties of disordered states and computational approaches generate representative ensembles of conformers using experimental restraints. In particular, NMR and small-angle X-ray scattering provide quantitative data that can readily be incorporated into calculations. These techniques have gleaned structural information about denatured, unfolded and intrinsically disordered proteins. The use of experimental data in different computational approaches, including ensemble molecular dynamics simulations and algorithms that assign populations to pregenerated conformers, has highlighted the presence of both local and long-range structure, and the occurrence of native-like and non-native interactions in unfolded and denatured states. Analysis of the resulting ensembles has suggested important implications of this fluctuating structure for folding, aggregation and binding.     

Does variation in host plant association and symbiont infection of pea aphid populations induce genetic and behaviour differentiation of its main parasitoid, Aphidius ervi?

The host-associated differentiation (HAD) hypothesis states that higher trophic levels in parasitic associations should exhibit similar divergence in case of host sympatric speciation. We tested HAD on populations of Aphidius ervi the main parasitoid of the pea aphid Acyrthosiphon pisum, emerging from host populations specialized on either alfalfa or red clover. Host and parasitoid populations were assessed for genetic variation and structure, while considering geography, host plant and host aphid protective symbionts Regiella insecticola and Hamiltonella defensa as potential covariables. Cluster and hierarchical analyses were used to assess the contribution of these variables to population structure, based on genotyping pea aphids and associated A. ervi with microsatellites, and host aphid facultative symbionts with 16S rDNA markers. Pea aphid genotypes were clearly distributed in two groups closely corresponding with their plant origins, confirming strong plant associated differentiation of this aphid in North America. Overall parasitism by A. ervi averaged 21.5 % across samples, and many parasitized aphids producing a wasp hosted defensive bacteria, indicating partial or ineffective protective efficacy of these symbionts in the field. The A. ervi population genetic data failed to support differentiation according to the host plant association of their pea aphid host. Potential for parasitoid specialization was also explored in experiments where wasps from alfalfa and clover aphids were reciprocally transplanted on alternate hosts, the hypothesis being that wasp behaviour and parasitic stages should be most adapted to their host of origin. Results revealed higher probability of oviposition on the alfalfa aphids, but higher adult emergence success on red clover aphids, with no interaction as expected under HAD. We conclude that our study provides no support for the HAD in this system. We discuss factors that might impair A. ervi specialization on its divergent aphid hosts on alfalfa and clover.     

Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability

Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.     

Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment

The protein-tyrosine phosphatase Shp2 plays an essential role in growth factor and integrin signaling, and Shp2 mutations cause developmental defects and/or malignancy. Previous work has placed Shp2 upstream of Ras. However, the mechanism of Shp2 action and its substrate(s) are poorly defined. Additional Shp2 functions downstream of, or parallel to, Ras/Erk activation also are proposed. Here, we show that Shp2 promotes Src family kinase (SFK) activation by regulating the phosphorylation of the Csk regulator PAG/Cbp, thereby controlling Csk access to SFKs. In Shp2-deficient cells, SFK inhibitory C-terminal tyrosines are hyperphosphorylated, and the tyrosyl phosphorylation of multiple SFK substrates, including Plcgamma1, is decreased. Decreased Plcgamma1 phosphorylation leads to defective Ras activation on endomembranes, and may help account for impaired Erk activation in Shp2-deficient cells. Decreased phosphorylation/activation of other SFK substrates may explain additional consequences of Shp2 deficiency, including altered cell spreading, stress fibers, focal adhesions, and motility.     

Chromosome 7 and 19 Trisomy in Cultured Human Neural Progenitor Cells

Background

Stem cell expansion and differentiation is the foundation of emerging cell therapy technologies. The potential applications of human neural progenitor cells (hNPCs) are wide ranging, but a normal cytogenetic profile is important to avoid the risk of tumor formation in clinical trials. FDA approved clinical trials are being planned and conducted for hNPC transplantation into the brain or spinal cord for various neurodegenerative disorders. Although human embryonic stem cells (hESCs) are known to show recurrent chromosomal abnormalities involving 12 and 17, no studies have revealed chromosomal abnormalities in cultured hNPCs. Therefore, we investigated frequently occurring chromosomal abnormalities in 21 independent fetal-derived hNPC lines and the possible mechanisms triggering such aberrations.

Methods and Findings

While most hNPC lines were karyotypically normal, G-band karyotyping and fluorescent in situ hybridization (FISH) analyses revealed the emergence of trisomy 7 (hNPC⁺⁷) and trisomy 19 (hNPC⁺¹⁹), in 24% and 5% of the lines, respectively. Once detected, subsequent passaging revealed emerging dominance of trisomy hNPCs. DNA microarray and immunoblotting analyses demonstrate epidermal growth factor receptor (EGFR) overexpression in hNPC⁺⁷ and hNPC⁺¹⁹ cells. We observed greater levels of telomerase (hTERT), increased proliferation (Ki67), survival (TUNEL), and neurogenesis (β_III-tubulin) in hNPC⁺⁷ and hNPC⁺¹⁹, using respective immunocytochemical markers. However, the trisomy lines underwent replicative senescence after 50–60 population doublings and never showed neoplastic changes. Although hNPC⁺⁷ and hNPC⁺¹⁹ survived better after xenotransplantation into the rat striatum, they did not form malignant tumors. Finally, EGF deprivation triggered a selection of trisomy 7 cells in a diploid hNPC line.

Conclusions

We report that hNPCs are susceptible to accumulation of chromosome 7 and 19 trisomy in long-term cell culture. These results suggest that micro-environmental cues are powerful factors in the selection of specific hNPC aneuploidies, with trisomy of chromosome 7 being the most common. Given that a number of stem cell based clinical trials are being conducted or planned in USA and a recent report in PLoS Medicine showing the dangers of grafting an inordinate number of cells, these data substantiate the need for careful cytogenetic evaluation of hNPCs (fetal or hESC-derived) before their use in clinical or basic science applications.