Functional Diversity and Structural Disorder in the Human Ubiquitination Pathway

The ubiquitin-proteasome system plays a central role in cellular regulation and protein quality control (PQC). The system is built as a pyramid of increasing complexity, with two E1 (ubiquitin activating), few dozen E2 (ubiquitin conjugating) and several hundred E3 (ubiquitin ligase) enzymes. By collecting and analyzing E3 sequences from the KEGG BRITE database and literature, we assembled a coherent dataset of 563 human E3s and analyzed their various physical features. We found an increase in structural disorder of the system with multiple disorder predictors (IUPred - E1: 5.97%, E2: 17.74%, E3: 20.03%). E3s that can bind E2 and substrate simultaneously (single subunit E3, ssE3) have significantly higher disorder (22.98%) than E3s in which E2 binding (multi RING-finger, mRF, 0.62%), scaffolding (6.01%) and substrate binding (adaptor/substrate recognition subunits, 17.33%) functions are separated. In ssE3s, the disorder was localized in the substrate/adaptor binding domains, whereas the E2-binding RING/HECT-domains were structured. To demonstrate the involvement of disorder in E3 function, we applied normal modes and molecular dynamics analyses to show how a disordered and highly flexible linker in human CBL (an E3 that acts as a regulator of several tyrosine kinase-mediated signalling pathways) facilitates long-range conformational changes bringing substrate and E2-binding domains towards each other and thus assisting in ubiquitin transfer. E3s with multiple interaction partners (as evidenced by data in STRING) also possess elevated levels of disorder (hubs, 22.90% vs. non-hubs, 18.36%). Furthermore, a search in PDB uncovered 21 distinct human E3 interactions, in 7 of which the disordered region of E3s undergoes induced folding (or mutual induced folding) in the presence of the partner. In conclusion, our data highlights the primary role of structural disorder in the functions of E3 ligases that manifests itself in the substrate/adaptor binding functions as well as the mechanism of ubiquitin transfer by long-range conformational transitions. © 2013 Bhowmick et al

Inflammasome sensor NLRP1 controls rat macrophage susceptibility to Toxoplasma gondii

Toxoplasma gondii is an intracellular parasite that infects a wide range of warm-blooded species. Rats vary in their susceptibility to this parasite. The Toxo1 locus conferring Toxoplasma resistance in rats was previously mapped to a region of chromosome 10 containing Nlrp1. This gene encodes an inflammasome sensor controlling macrophage sensitivity to anthrax lethal toxin (LT) induced rapid cell death (pyroptosis). We show here that rat strain differences in Toxoplasma infected macrophage sensitivity to pyroptosis, IL-1β/IL-18 processing, and inhibition of parasite proliferation are perfectly correlated with NLRP1 sequence, while inversely correlated with sensitivity to anthrax LT-induced cell death. Using recombinant inbred rats, SNP analyses and whole transcriptome gene expression studies, we narrowed the candidate genes for control of Toxoplasma-mediated rat macrophage pyroptosis to four genes, one of which was Nlrp1. Knockdown of Nlrp1 in pyroptosis-sensitive macrophages resulted in higher parasite replication and protection from cell death. Reciprocally, overexpression of the NLRP1 variant from Toxoplasma-sensitive macrophages in pyroptosis-resistant cells led to sensitization of these resistant macrophages. Our findings reveal Toxoplasma as a novel activator of the NLRP1 inflammasome in rat macrophages

Quantitative comparison of myocardial fiber structure between mice, rabbit, and sheep using diffusion tensor cardiovascular magnetic resonance

<p>Abstract</p> <p>Background</p> <p>Accurate interpretations of cardiac functions require precise structural models of the myocardium, but the latter is not available always and for all species. Although scaling or substitution of myocardial fiber information from alternate species has been used in cardiac functional modeling, the validity of such practice has not been tested.</p> <p>Methods</p> <p>Fixed mouse (n = 10), rabbit (n = 6), and sheep (n = 5) hearts underwent diffusion tensor imaging (DTI). The myocardial structures in terms of the left ventricular fiber orientation helix angle index were quantitatively compared between the mouse rabbit and sheep hearts.</p> <p>Results</p> <p>The results show that significant fiber structural differences exist between any two of the three species. Specifically, the subepicardial fiber orientation, and the transmural range and linearity of fiber helix angles are significantly different between the mouse and either rabbit or sheep. Additionally, a significant difference was found between the transmural helix angle range between the rabbit and sheep. Across different circumferential regions of the heart, the fiber orientation was not found to be significantly different.</p> <p>Conclusions</p> <p>The current study indicates that myocardial structural differences exist between different size hearts. An immediate implication of the present findings for myocardial structural or functional modeling studies is that caution must be exercised when extrapolating myocardial structures from one species to another.</p

Heterogeneity of fractional anisotropy and mean diffusivity measurements by in vivo diffusion tensor imaging in normal human hearts

Background: Cardiac diffusion tensor imaging (cDTI) by cardiovascular magnetic resonance has the potential to assess microstructural changes through measures of fractional anisotropy (FA) and mean diffusivity (MD). However, normal variation in regional and transmural FA and MD is not well described. Methods: Twenty normal subjects were scanned using an optimised cDTI sequence at 3T in systole. FA and MD were quantified in 3 transmural layers and 4 regional myocardial walls. Results: FA was higher in the mesocardium (0.46 ±0.04) than the endocardium (0.40 ±0.04, p≤0.001) and epicardium (0.39 ±0.04, p≤0.001). On regional analysis, the FA in the septum was greater than the lateral wall (0.44 ±0.03 vs 0.40 ±0.05 p = 0.04). There was a transmural gradient in MD increasing towards the endocardium (epicardium 0.87 ±0.07 vs endocardium 0.91 ±0.08×10-3 mm2/s, p = 0.04). With the lateral wall (0.87 ± 0.08×10-3 mm2/s) as the reference, the MD was higher in the anterior wall (0.92 ±0.08×10-3 mm2/s, p = 0.016) and septum (0.92 ±0.07×10-3 mm2/s, p = 0.028). Transmurally the signal to noise ratio (SNR) was greatest in the mesocardium (14.5 ±2.5 vs endocardium 13.1 ±2.2, p<0.001; vs epicardium 12.0 ± 2.4, p<0.001) and regionally in the septum (16.0 ±3.4 vs lateral wall 11.5 ± 1.5, p<0.001). Transmural analysis suggested a relative reduction in the rate of change in helical angle (HA) within the mesocardium. Conclusions: In vivo FA and MD measurements in normal human heart are heterogeneous, varying significantly transmurally and regionally. Contributors to this heterogeneity are many, complex and interactive, but include SNR, variations in cardiac microstructure, partial volume effects and strain. These data indicate that the potential clinical use of FA and MD would require measurement standardisation by myocardial region and layer, unless pathological changes substantially exceed the normal variation identified

α-Adducin Gly460Trp Gene Mutation and Essential Hypertension in a Chinese Population: A Meta-Analysis including 10960 Subjects

BACKGROUND: The α-adducin Gly460Trp (G460W) gene polymorphism may be associated with susceptibility to essential hypertension (EH), but this relationship remains controversial. In an attempt to resolve this issue, we conducted a meta-analysis. METHODS: Twenty-three separated studies involving 5939 EH patients and 5021 controls were retrieved and analyzed. Four ethnicities were included: Han, Kazakh, Mongolian, and She. Eighteen studies with 5087 EH patients and 4183 controls were included in the Han subgroup. Three studies with 636 EH patients and 462 controls were included in the Kazakh subgroup. The Mongolian subgroup was represented by only one study with 100 EH patients and 50 controls; similarly, only one study with 116 EH patients and 326 controls was available for the She subgroup. The pooled and ethnic group odds ratios (ORs) along with the corresponding 95% confidence intervals (95% CI) were assessed using a random effects model. RESULTS: There was a significant association between the α-adducin G460W gene polymorphism and EH in the pooled Chinese population under both an allelic genetic model (OR: 1.12, 95% CI: 1.04-1.20, P = 0.002) and a recessive genetic model (OR: 1.40, 95% CI: 1.16-1.70, P = 0.0005). In contrast, no significant association between the α-adducin G460W gene polymorphism and EH was observed in the dominant genetic model (OR: 0.88, 95% CI: 0.72-1.09, P = 0.24). In stratified analysis by ethnicity, significantly increased risk was detected in the Han subgroup under an allelic genetic model (OR: 1.13, 95% CI: 1.04-1.23, P = 0.003) and a recessive genetic model (OR: 1.43, 95% CI: 1.17-1.75, P = 0.0006). CONCLUSIONS: In a Chinese population of mixed ethnicity, the α-adducin G460W gene polymorphism was linked to EH susceptibility, most strongly in Han Chinese

Intrinsically Disordered Proteins Display No Preference for Chaperone Binding In Vivo

Intrinsically disordered/unstructured proteins (IDPs) are extremely sensitive to proteolysis in vitro, but show no enhanced degradation rates in vivo. Their existence and functioning may be explained if IDPs are preferentially associated with chaperones in the cell, which may offer protection against degradation by proteases. To test this inference, we took pairwise interaction data from high-throughput interaction studies and analyzed to see if predicted disorder correlates with the tendency of chaperone binding by proteins. Our major finding is that disorder predicted by the IUPred algorithm actually shows negative correlation with chaperone binding in E. coli, S. cerevisiae, and metazoa species. Since predicted disorder positively correlates with the tendency of partner binding in the interactome, the difference between the disorder of chaperone-binding and non-binding proteins is even more pronounced if normalized to their overall tendency to be involved in pairwise protein–protein interactions. We argue that chaperone binding is primarily required for folding of globular proteins, as reflected in an increased preference for chaperones of proteins in which at least one Pfam domain exists. In terms of the functional consequences of chaperone binding of mostly disordered proteins, we suggest that its primary reason is not the assistance of folding, but promotion of assembly with partners. In support of this conclusion, we show that IDPs that bind chaperones also tend to bind other proteins

Effects of Supplementary Feeding on the Breeding Ecology of the Buff-Throated Partridge in a Tibetan Sacred Site, China

Our goal was to document effects of year-round supplemental feeding on breeding ecology of the Buff-throated Partridge, Tetraophasis szechenyii, within a Tibetan sacred site. We evaluated effects of supplemental feeding used as religious/cultural practices which could potentially aid conservation of endangered phasianids. We compared fed breeding groups to neighboring nonfed groups. Fed groups initiated first clutches significantly earlier than nonfed groups. Earlier laying groups within fed and nonfed groups showed significantly lower hatching rates than later groups; however, fed groups showed significantly higher hatching rates than nonfed groups laying in the same period. Earlier laying increased opportunities to renest. All six fed groups with clutch failures renested compared to only one of five nonfed groups with clutch failures. Fed female breeders showed significantly greater investment in their young with larger clutches and larger eggs, which likely increased survivability of early hatchlings. We observed no predation on birds at feeding sites and recorded only four cases of predation on incubating females, which showed no detectable difference between fed and nonfed groups. Ground-nesting birds typically face high risks of predation. Ten of the 48 groups nested in trees, which occurs in few phasianid species. Tree nests showed significantly higher hatching rates compared to ground nests; however, we found no significant difference in tree nesting between fed and nonfed groups. This partridge is one of four gallinaceous species with cooperative breeding. Breeding groups with helpers had significantly greater reproductive success than single pairs, and fed female breeders with helpers laid bigger eggs than single pairs. Comparing annual reproductive output per group, fed groups not only produced significantly more independent young (≥150 days post-hatching), their young hatched significantly earlier, which likely have greater reproductive value over later hatched young of nonfed groups. Supplemental feeding year-round is likely what enabled the successes of the fed partridges