Most studies of protein networks operate on a high level of abstraction, neglecting structural and chemical aspects of each interaction. Here, we characterize interactions by using atomic-resolution ...information from three-dimensional protein structures. We find that some previously recognized relationships between network topology and genomic features (e.g., hubs tending to be essential proteins) are actually more reflective of a structural quantity, the number of distinct binding interfaces. Subdividing hubs with respect to this quantity provides insight into their evolutionary rate and indicates that additional mechanisms of network growth are active in evolution (beyond effective preferential attachment through gene duplication).
Eukaryotic protein kinases are generally classified as being either tyrosine or serine-threonine specific. Though not evident from inspection of their primary sequences, many serine-threonine kinases ...display a significant preference for serine or threonine as the phosphoacceptor residue. Here we show that a residue located in the kinase activation segment, which we term the “DFG+1” residue, acts as a major determinant for serine-threonine phosphorylation site specificity. Mutation of this residue was sufficient to switch the phosphorylation site preference for multiple kinases, including the serine-specific kinase PAK4 and the threonine-specific kinase MST4. Kinetic analysis of peptide substrate phosphorylation and crystal structures of PAK4-peptide complexes suggested that phosphoacceptor residue preference is not mediated by stronger binding of the favored substrate. Rather, favored kinase-phosphoacceptor combinations likely promote a conformation optimal for catalysis. Understanding the rules governing kinase phosphoacceptor preference allows kinases to be classified as serine or threonine specific based on their sequence.
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•A single active site residue can determine kinase phosphoacceptor specificity•Favored and disfavored substrates promote distinct kinase-bound conformations•A simple rule predicts kinase phosphoacceptor preference from its DFG+1 residue
The Krebs cycle-derived metabolite itaconate and its derivatives suppress the inflammatory response in pro-inflammatory “M1” macrophages. However, alternatively activated “M2” macrophages can take up ...itaconate. We therefore examined the effect of itaconate and 4-octyl itaconate (OI) on M2 macrophage activation. We demonstrate that itaconate and OI inhibit M2 polarization and metabolic remodeling. Examination of IL-4 signaling revealed inhibition of JAK1 and STAT6 phosphorylation by both itaconate and OI. JAK1 activation was also inhibited by OI in response to IL-13, interferon-β, and interferon-γ in macrophages and in T helper 2 (Th2) cells. Importantly, JAK1 was directly modified by itaconate derivatives at multiple residues, including cysteines 715, 816, 943, and 1130. Itaconate and OI also inhibited JAK1 kinase activity. Finally, OI treatment suppressed M2 macrophage polarization and JAK1 phosphorylation in vivo. We therefore identify itaconate and OI as JAK1 inhibitors, suggesting a new strategy to inhibit JAK1 in M2 macrophage-driven diseases.
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•Itaconate reduces M2 macrophage polarization, JAK1/STAT6 activation, and OXPHOS•Itaconate derivative OI is a JAK1 inhibitor in type 1 and 2 cytokine pathways•Cysteine residues on JAK1 are directly modified by itaconate derivates•Itaconate derivative OI inhibits JAK phosphorylation and asthma severity in vivo
The wide-reaching roles of the immunomodulatory metabolite itaconate are incompletely understood. Here, Runtsch et al. demonstrate that itaconate and derivatives directly modify JAK1 and are JAK1 inhibitors during macrophage alternative activation. These discoveries provide a novel basis for itaconate and derivatives as therapies in type-2-driven diseases, including asthma.
SH3 domains are peptide recognition modules that mediate the assembly of diverse biological complexes. We scanned billions of phage-displayed peptides to map the binding specificities of the SH3 ...domain family in the budding yeast, Saccharomyces cerevisiae. Although most of the SH3 domains fall into the canonical classes I and II, each domain utilizes distinct features of its cognate ligands to achieve binding selectivity. Furthermore, we uncovered several SH3 domains with specificity profiles that clearly deviate from the two canonical classes. In conjunction with phage display, we used yeast two-hybrid and peptide array screening to independently identify SH3 domain binding partners. The results from the three complementary techniques were integrated using a Bayesian algorithm to generate a high-confidence yeast SH3 domain interaction map. The interaction map was enriched for proteins involved in endocytosis, revealing a set of SH3-mediated interactions that underlie formation of protein complexes essential to this biological pathway. We used the SH3 domain interaction network to predict the dynamic localization of several previously uncharacterized endocytic proteins, and our analysis suggests a novel role for the SH3 domains of Lsb3p and Lsb4p as hubs that recruit and assemble several endocytic complexes.
Providing equitable care is an objective of many national healthcare systems. Using the birth cohort of the nationally representative Longitudinal Study of Australian Children linked with the ...Medicare Benefits Scheme billing data who were recruited in 2004 at ages 0–1 years and assessed biennially for six waves, we assessed the distribution of out-of-hospital government Medicare spending by household income. 4853 children followed over 11 years were included in the study. Distributions of major spending components including general practitioner and specialist care were assessed using concentration indices. Trends in the inequalities as children grow were investigated. The results showed that after controlling for health care needs, total government Medicare spending over 0–11 years of age favoured the rich (concentration index 0.041). The Medicare spending for general practitioner care was equal (concentration index 0.005) while for specialist care and diagnostics and imaging were ‘pro-rich’ (concentration index 0.108 and 0.088 respectively). Children from poorer families were most disadvantaged when aged 0–1 years in specialist spending, and the disparity lessened as children approached adolescence. Our findings suggest that income-related inequalities exist in government Medicare spending particularly in the first few years of life. As early years of life are a critical window in childhood development and building block for future health, the results warrant further investigation and attention from policy makers.
•Medicare GP spending for children was neutral regarding income.•Children from lower income families received less specialist care spending.•Income-related inequality in specialist spending was most profound early in life.•The disparity lessened as children approached adolescence.
Structural variation of the genome involves kilobase- to megabase-sized deletions, duplications, insertions, inversions, and complex combinations of rearrangements. We introduce high-throughput and ...massive paired-end mapping (PEM), a large-scale genome-sequencing method to identify structural variants (SVs) ~3 kilobases (kb) or larger that combines the rescue and capture of paired ends of 3-kb fragments, massive 454 sequencing, and a computational approach to map DNA reads onto a reference genome. PEM was used to map SVs in an African and in a putatively European individual and identified shared and divergent SVs relative to the reference genome. Overall, we fine-mapped more than 1000 SVs and documented that the number of SVs among humans is much larger than initially hypothesized; many of the SVs potentially affect gene function. The breakpoint junction sequences of more than 200 SVs were determined with a novel pooling strategy and computational analysis. Our analysis provided insights into the mechanisms of SV formation in humans.
Severe, steroid-resistant asthma is the major unmet need in asthma therapy. Disease heterogeneity and poor understanding of pathogenic mechanisms hampers the identification of therapeutic targets. ...Excessive nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome and concomitant IL-1β responses occur in chronic obstructive pulmonary disease, respiratory infections, and neutrophilic asthma. However, the direct contributions to pathogenesis, mechanisms involved, and potential for therapeutic targeting remain poorly understood, and are unknown in severe, steroid-resistant asthma.
To investigate the roles and therapeutic targeting of the NLRP3 inflammasome and IL-1β in severe, steroid-resistant asthma.
We developed mouse models of Chlamydia and Haemophilus respiratory infection-mediated, ovalbumin-induced severe, steroid-resistant allergic airway disease. These models share the hallmark features of human disease, including elevated airway neutrophils, and NLRP3 inflammasome and IL-1β responses. The roles and potential for targeting of NLRP3 inflammasome, caspase-1, and IL-1β responses in experimental severe, steroid-resistant asthma were examined using a highly selective NLRP3 inhibitor, MCC950; the specific caspase-1 inhibitor Ac-YVAD-cho; and neutralizing anti-IL-1β antibody. Roles for IL-1β-induced neutrophilic inflammation were examined using IL-1β and anti-Ly6G.
Chlamydia and Haemophilus infections increase NLRP3, caspase-1, IL-1β responses that drive steroid-resistant neutrophilic inflammation and airway hyperresponsiveness. Neutrophilic airway inflammation, disease severity, and steroid resistance in human asthma correlate with NLRP3 and IL-1β expression. Treatment with anti-IL-1β, Ac-YVAD-cho, and MCC950 suppressed IL-1β responses and the important steroid-resistant features of disease in mice, whereas IL-1β administration recapitulated these features. Neutrophil depletion suppressed IL-1β-induced steroid-resistant airway hyperresponsiveness.
NLRP3 inflammasome responses drive experimental severe, steroid-resistant asthma and are potential therapeutic targets in this disease.
Purpose
Resistance to endocrine therapy is the primary cause of treatment failure and death in patients with ER-positive (ER +)/luminal breast cancer. Expression and activation of the RET receptor ...tyrosine kinase may be driving poor outcomes. We aim to identify high-risk patients and druggable pathways for biomarker-based clinical trials.
Methods
We obtained batch-normalized mRNA expression data from Breast Invasive Carcinoma-The Cancer Genome Atlas, PanCancer Atlas (BRCA-TCGA). To determine clinically significant cutoffs for RET expression, patients were grouped at different thresholds for Kaplan–Meier plotting. Differential gene expression (DGE) analysis and enrichment for gene sets was performed. transcriptomic dataset of antiestrogen-treated ER + tumors stratified by clinical response was then analyzed.
Results
High RET expression was associated with worse outcomes in patients with ER + tumors, and stratification was enhanced by incorporating GDNF expression. High RET/GDNF patients had significantly lower overall survival (HR = 2.04,
p
= 0.012), progression-free survival (HR = 2.87,
p
< 0.001), disease-free survival (HR = 2.67,
p
< 0.001), and disease-specific survival (HR = 3.53,
p
< 0.001) than all other ER + patients. High RET/GDNF tumors were enriched for estrogen-independent signaling and targetable pathways including NTRK, PI3K, and KRAS. Tumors with adaptive resistance to endocrine therapy were enriched for gene expression signatures of high RET/GDNF primary tumors.
Conclusion
Expression and activation of the RET receptor tyrosine kinase may be driving poor outcomes in some patients with ER + breast cancer. ER + patients above the 75th percentile may benefit from clinical trials with tyrosine kinase inhibitors.
ELASPIC is a novel ensemble machine-learning approach that predicts the effects of mutations on protein folding and protein-protein interactions. Here, we present the ELASPIC webserver, which makes ...the ELASPIC pipeline available through a fast and intuitive interface. The webserver can be used to evaluate the effect of mutations on any protein in the Uniprot database, and allows all predicted results, including modeled wild-type and mutated structures, to be managed and viewed online and downloaded if needed. It is backed by a database which contains improved structural domain definitions, and a list of curated domain-domain interactions for all known proteins, as well as homology models of domains and domain-domain interactions for the human proteome. Homology models for proteins of other organisms are calculated on the fly, and mutations are evaluated within minutes once the homology model is available.
The ELASPIC webserver is available online at http://elaspic.kimlab.org
pm.kim@utoronto.ca or pi@kimlab.orgSupplementary data: Supplementary data are available at Bioinformatics online.
Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits its speed. We ...developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and computationally recovers high-resolution images, attaining voxel rates of over 1 billion Hz in structured samples. Using a static image as a prior for recording neural activity, we imaged visually evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 µm and frame rates over 1 kHz. Dendritic glutamate transients in anesthetized mice are synchronized within spatially contiguous domains spanning tens of micrometers at frequencies ranging from 1-100 Hz. We demonstrate millisecond-resolved recordings of acetylcholine and voltage indicators, three-dimensional single-particle tracking and imaging in densely labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.
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