Germline disease-causing variants are generally more spatially clustered in protein 3-dimensional structures than benign variants. Motivated by this tendency, we develop a fast and powerful ...protein-structure-based scan (PSCAN) approach for evaluating gene-level associations with complex disease and detecting signal variants. We validate PSCAN's performance on synthetic data and two real data sets for lipid traits and Alzheimer's disease. Our results demonstrate that PSCAN performs competitively with existing gene-level tests while increasing power and identifying more specific signal variant sets. Furthermore, PSCAN enables generation of hypotheses about the molecular basis for the associations in the context of protein structures and functional domains.
We report a genome-wide RNA interference (RNAi) screen for Suppressors of Clozapine-induced Larval Arrest (scla genes) in Caenorhabditis elegans, the first genetic suppressor screen for antipsychotic ...drug (APD) targets in an animal. The screen identifies 40 suppressors, including the α-like nicotinic acetylcholine receptor (nAChR) homolog acr-7. We validate the requirement for acr-7 by showing that acr-7 knockout suppresses clozapine-induced larval arrest and that expression of a full-length translational GFP fusion construct rescues this phenotype. nAChR agonists phenocopy the developmental effects of clozapine, while nAChR antagonists partially block these effects. ACR-7 is strongly expressed in the pharynx, and clozapine inhibits pharyngeal pumping. acr-7 knockout and nAChR antagonists suppress clozapine-induced inhibition of pharyngeal pumping. These findings suggest that clozapine activates ACR-7 channels in pharyngeal muscle, leading to tetanus of pharyngeal muscle with consequent larval arrest. No APDs are known to activate nAChRs, but a number of studies indicate that α7-nAChR agonists may prove effective for the treatment of psychosis. α-like nAChR signaling is a mechanism through which clozapine may produce its therapeutic and/or toxic effects in humans, a hypothesis that could be tested following identification of the mammalian ortholog of C. elegans acr-7.
Structural characterization of the human Y4 receptor (hY4R) interaction with human pancreatic polypeptide (hPP) is crucial, not only for understanding its biological function but also for testing ...treatment strategies for obesity that target this interaction. Here, the interaction of receptor mutants with pancreatic polypeptide analogs was studied through double-cycle mutagenesis. To guide mutagenesis and interpret results, a three-dimensional comparative model of the hY4R-hPP complex was constructed based on all available class A G protein-coupled receptor crystal structures and refined using experimental data. Our study reveals that residues of the hPP and the hY4R form a complex network consisting of ionic interactions, hydrophobic interactions, and hydrogen binding. Residues Tyr2.64, Asp2.68, Asn6.55, Asn7.32, and Phe7.35 of Y4R are found to be important in receptor activation by hPP. Specifically, Tyr2.64 interacts with Tyr27 of hPP through hydrophobic contacts. Asn7.32 is affected by modifications on position Arg33 of hPP, suggesting a hydrogen bond between these two residues. Likewise, we find that Phe7.35 is affected by modifications of hPP at positions 33 and 36, indicating interactions between these three amino acids. Taken together, we demonstrate that the top of transmembrane helix 2 (TM2) and the top of transmembrane helices 6 and 7 (TM6–TM7) form the core of the peptide binding pocket. These findings will contribute to the rational design of ligands that bind the receptor more effectively to produce an enhanced agonistic or antagonistic effect.
The Y4R is involved in regulation of food intake and gastrointestinal transport.
Mutagenesis studies revealed several residues displaying a significant loss of potency for hPP.
Tops of TM2, TM6, and TM7 interact with the hY4R native agonist hPP.
Characterizing the structure of the Y4R binding pocket is crucial for the development of new anti-obesity drugs.
Immunity genes have repeatedly experienced natural selection during mammalian evolution. Galectins are carbohydrate-binding proteins that regulate diverse immune responses, including maternal-fetal ...immune tolerance in placental pregnancy. Seven human galectins, four conserved across vertebrates and three specific to primates, are involved in placental development. To comprehensively study the molecular evolution of these galectins, both across mammals and within humans, we conducted a series of between- and within-species evolutionary analyses. By examining patterns of sequence evolution between species, we found that primate-specific galectins showed uniformly high substitution rates, whereas two of the four other galectins experienced accelerated evolution in primates. By examining human population genomic variation, we found that galectin genes and variants, including variants previously linked to immune diseases, showed signatures of recent positive selection in specific human populations. By examining one nonsynonymous variant in Galectin-8 previously associated with autoimmune diseases, we further discovered that it is tightly linked to three other nonsynonymous variants; surprisingly, the global frequency of this four-variant haplotype is ∼50%. To begin understanding the impact of this major haplotype on Galectin-8 protein structure, we modeled its 3D protein structure and found that it differed substantially from the reference protein structure. These results suggest that placentally expressed galectins experienced both ancient and more recent selection in a lineage- and population-specific manner. Furthermore, our discovery that the major Galectin-8 haplotype is structurally distinct from and more commonly found than the reference haplotype illustrates the significance of understanding the evolutionary processes that sculpted variants associated with human genetic disease.
Clozapine has superior and unique effects as an antipsychotic agent, but the mediators of these effects are not known. We studied behavioral and developmental effects of clozapine in Caenorhabditis ...elegans, as a model system to identify previously undiscovered mechanisms of drug action. Clozapine induced early larval arrest, a phenotype that was also seen with the clozapine metabolite N-desmethyl clozapine but not with any other typical or atypical antipsychotic drug tested. Mutations in the insulin receptor/daf-2 and phosphatidyl inositol 3-kinase (PI3K)/age-1 suppressed clozapine-induced larval arrest, suggesting that clozapine may activate the insulin-signaling pathway. Consistent with this notion, clozapine also increased the expression of an age-1::GFP reporter. Activation of the insulin-signaling pathway leads to cytoplasmic localization of the fork head transcription factor FOXO/daf-16. Clozapine produced cytoplasmic localization of DAF-16::GFP in arrested L1 larvae, in contrast to stressors such as starvation or high temperature, which produce nuclear localization of DAF-16::GFP in arrested L1 larvae. Clozapine also inhibited pharyngeal pumping in C. elegans, an effect that may contribute to, but did not explain, clozapine-induced larval arrest. Our findings demonstrate a drug-specific interaction between clozapine and the PI3K/insulin-signaling pathway in C. elegans. As this pathway is conserved across species, the results may have implications for understanding the unique effects of clozapine in humans.
We report a
gatekeeper mutation in a patient with
-mutant breast cancer with acquired resistance to neratinib. Laboratory studies suggested that
is a neratinib-sensitive, gain-of-function mutation ...that upon dimerization with mutant HER3
, also present in the breast cancer, amplifies HER2 signaling. The patient was treated with neratinib and exhibited a sustained partial response. Upon clinical progression,
was detected in plasma tumor cell-free DNA. Structural modeling of this acquired mutation suggested that the increased bulk of isoleucine in HER2
reduces neratinib binding. Neratinib blocked HER2-mediated signaling and growth in cells expressing HER2
but not HER2
In contrast, afatinib and the osimertinib metabolite AZ5104 strongly suppressed HER2
-induced signaling and cell growth. Acquisition of HER2
upon development of resistance to neratinib in a breast cancer with an initial activating
mutation suggests
is a driver mutation. HER2
-mediated neratinib resistance may be overcome by other irreversible HER2 inhibitors like afatinib.
We found an acquired
gatekeeper mutation in a patient with
-mutant breast cancer upon clinical progression on neratinib. We speculate that
may arise as a secondary mutation following response to effective HER2 tyrosine kinase inhibitors (TKI) in other cancers with
-activating mutations. This resistance may be overcome by other irreversible HER2 TKIs, such as afatinib.
.
PDF
