Elucidating substrate and inhibitor binding sites on the surface of GSK-3β and the refinement of a competitive inhibitor

A molecular understanding of substrate recognition of protein kinases provides an important basis for the development of substrate competitive inhibitors. Here, we explored substrate recognition and competitive inhibition of glycogen synthase kinase (GSK)-3β using molecular and computational tools. In previous work, we described Gln89 and Asn95 within GSK-3β as important substrates binding sites. Here, we show that the cavity bordered by loop 89-QDKRFKN-95, located in the vicinity of the GSK-3β catalytic core, is a promiscuous substrate binding subsite. Mutations within this segment highlighted Phe93 as an additional essential contact residue for substrates' recognition. However, unlike Gln89 and Asn95, Phe93 was also important for the binding of our previously described substrate competitive inhibitor, L803 [KEAPPAPPQS(p)P], and its cell-permeable variant L803-mts. The effects of the substitution of charged or polar residues within L803 further suggested that binding to GSK-3β is governed by hydrophobic interactions. Our computational model of GSK-3β bound to L803 was in agreement with the experimental data. It revealed L803 binding with a hydrophobic surface patch and identified interactions between Pro8 (L803) and Phe93 (GSK-3β). Computational modeling of new L803 variants predicted that inhibition would be strengthened by adding contacts with Phe93 or by increasing the hydrophobic content of the peptide. Indeed, the newly designed L803 variants showed improved inhibition. Our study identified different and overlapping elements in GSK-3β substrate and inhibitor recognition and provides a novel example for model-based rational design of substrate competitive inhibitors for GSK-3.     

The Popdc gene family in the rat: molecular cloning, characterization and expression analysis in the heart and cultured cardiomyocytes

Three Popeye domain-containing (Popdc 1-3) family-members are known in vertebrates. Their exact function is as yet unknown although involvement in cell adhesion has been suggested. We report herein sequencing of the rat Popdc 1-3 cDNAs that show high homology to other vertebrate orthologs and are expressed primarily in the heart and skeletal muscles. Popdc2 splice variants were identified, with Popdc2C showing a distinctive age-dependent decline. In isolated cardiomyocytes, Popdc genes were negatively regulated by serum, an effect that was reversed by EGFR-kinase inhibition, suggesting an EGFR-dependent modulation of Popdc gene expression.     

Seeing is believing: new methods for in situ single-cell transcriptomics

New methods employ RNA-seq to study single cells within complex tissues by in situ sequencing or mRNA capture from single photoactivated cells.     

PKD at the crossroads of necrosis and autophagy

Reactive oxygen species (ROS) that accumulate under oxidative pressure cause severe damage to cellular components, and induce various cellular responses, including apoptosis, programmed necrosis and autophagy, depending on the cellular setting. Various studies have described ROS-induced autophagy, but only a few direct factors that regulate autophagy under oxidative stress are known to date. We have identified DAPK and PKD as such regulators by demonstrating their role in the process of autophagy in general, and specifically during oxidative stress. PKD acts as a downstream effector of DAPk in the regulation of autophagy. Furthermore, PKD functions within the autophagic network as an activator of VPS34, by associating with and phosphorylating VPS34, leading to its activation. Significantly, PKD is recruited to the autophagosomal membranes, placing it within proximity of its autophagic target.     

Global scale variation in the salinity sensitivity of riverine macroinvertebrates: eastern Australia, France, Israel and South Africa

Salinity is a key abiotic property of inland waters; it has a major influence on biotic communities and is affected by many natural and anthropogenic processes. Salinity of inland waters tends to increase with aridity, and biota of inland waters may have evolved greater salt tolerance in more arid regions. Here we compare the sensitivity of stream macroinvertebrate species to salinity from a relatively wet region in France (Lorraine and Brittany) to that in three relatively arid regions eastern Australia (Victoria, Queensland and Tasmania), South Africa (south-east of the Eastern Cape Province) and Israel using the identical experimental method in all locations. The species whose salinity tolerance was tested, were somewhat more salt tolerant in eastern Australia and South Africa than France, with those in Israel being intermediate. However, by far the greatest source of variation in species sensitivity was between taxonomic groups (Order and Class) and not between the regions. We used a bayesian statistical model to estimate the species sensitivity distributions (SSDs) for salinity in eastern Australia and France adjusting for the assemblages of species in these regions. The assemblage in France was slightly more salinity sensitive than that in eastern Australia. We therefore suggest that regional salinity sensitivity is therefore likely to depend most on the taxonomic composition of respective macroinvertebrate assemblages. On this basis it would be possible to screen rivers globally for risk from salinisation.     

Cutting edge: MHC class I-Ly49 interaction regulates neuronal function

MHC class I molecules (MHC-I) have been implicated in nervous system development in the mouse. In this study we present evidence for the interaction of MHC-I with the NK cell receptor Ly49 in primary cortical neuronal cultures. We show that MHC-I and Ly49 are expressed on neuronal soma and axon surfaces, with Ly49 also present on dendrites. Anti-MHC-I Abs reduce synapsin-I expression and enhance neurite outgrowth and neuronal death. Conversely, anti-Ly49 mAbs increase synapsin-I expression, reduce neurite outgrowth, and promote neuron viability. Because we show that Ly49 genes are selectively expressed in the adult brain, these findings suggest an unsuspected role for the MHC-I-Ly49 interaction in the development and function of the brain.     

Serum histones as biomarkers of the severity of heatstroke in dogs

Heatstroke is associated with systemic inflammatory response syndrome, leading to multiple organ dysfunction and death. Currently, there is no specific treatment decreasing hyperthermia-induced inflammatory/hemostatic derangements. Emerging studies indicate that histones leaking from damaged cells into the extracellular space are toxic, pro-inflammatory, and pro-thrombotic. We therefore hypothesize that serum histones (sHs) are elevated during heatstroke and are associated with the severity of the disease. Sixteen dogs with heatstroke and seven healthy controls were included in the study. Median serum histones (sHs) upon admission in dogs with heatstroke were significantly higher (P = 0.043) compared to that in seven controls (13.2 vs. 7.3 ng/mL, respectively). sHs level was significantly higher among non-survivors and among dogs with severe hemostatic derangement (P = 0.049, median 21.4 ng/mL vs. median 8.16 ng/mL and P = 0.038, 19.0 vs. 7.0 ng/mL, respectively). There were significant positive correlation between sHs and urea (r = 0.8, P = 0.02); total CO2 (r = 0.661, P = 0.05); CK (r = 0.678, P = 0.04); and prothrombin time (PT) 12 h post presentation (r = 0.888, P = 0.04). The significant positive correlation between sHs and other heatstroke severity biomarkers, and significant increase among severely affected dogs, implies its role in inflammation/oxidation/coagulation during heatstroke. sHs, unlike other prognostic and severity biomarkers in heatstroke, can be pharmacologically manipulated, offering a potential therapeutic target.     

Characterization of coding synonymous and non-synonymous variants in ADAMTS13 using ex vivo and in silico approaches

Synonymous variations, which are defined as codon substitutions that do not change the encoded amino acid, were previously thought to have no effect on the properties of the synthesized protein(s). However, mounting evidence shows that these "silent" variations can have a significant impact on protein expression and function and should no longer be considered "silent". Here, the effects of six synonymous and six non-synonymous variations, previously found in the gene of ADAMTS13, the von Willebrand Factor (VWF) cleaving hemostatic protease, have been investigated using a variety of approaches. The ADAMTS13 mRNA and protein expression levels, as well as the conformation and activity of the variants have been compared to that of wild-type ADAMTS13. Interestingly, not only the non-synonymous variants but also the synonymous variants have been found to change the protein expression levels, conformation and function. Bioinformatic analysis of ADAMTS13 mRNA structure, amino acid conservation and codon usage allowed us to establish correlations between mRNA stability, RSCU, and intracellular protein expression. This study demonstrates that variants and more specifically, synonymous variants can have a substantial and definite effect on ADAMTS13 function and that bioinformatic analysis may allow development of predictive tools to identify variants that will have significant effects on the encoded protein.     

Resistance, resilience, and community dynamics in mediterranean-climate streams

Streams and rivers in mediterranean-climate regions (med-rivers) are subjected to sequential, yet contrasting hydrologic disturbances of drying and flooding. Although seasonally predictable, these disturbances can vary in intensity and duration within and among mediterranean-climate regions (med-regions). Consequently, med-rivers differ in the permanence of their aquatic habitats. To persist, species have acquired matched resistance and resilience adaptations. They gain resistance either by enduring the stress or avoiding it. Community recovery (or resilience) is achieved with cessation of hydrologic stress that permits maximization of re-colonization and reproduction. Endurance strategies are usually disturbance-specific, but avoidance enables organisms to cope with both drying and flooding, and is the prevalent resistance strategy. Correspondingly, community persistence depends to a large extent on the integrity of refuges, an aspect that has so far been little explored. Existing information suggests that seasonal community succession becomes more pronounced with increasing aridity and declining water permanence. The invertebrate community in semi-arid med-rivers can therefore undergo succession through three to four identifiable assemblages, whereas in perennial streams the difference between wet and dry period assemblages is smaller. Community turnover is influenced by the intensity of the hydrologic disturbances and varies between wet and drought years.