Pharmacological responses of G protein-coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR ...structures. We reengineered the human A 2A adenosine receptor by replacing its third intracellular loop with apocytochrome b⁵⁶² RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp 2.50 . Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.
The 9p21.3 cardiovascular disease locus is the most influential common genetic risk factor for coronary artery disease (CAD), accounting for ∼10%–15% of disease in non-African populations. The ∼60 kb ...risk haplotype is human-specific and lacks coding genes, hindering efforts to decipher its function. Here, we produce induced pluripotent stem cells (iPSCs) from risk and non-risk individuals, delete each haplotype using genome editing, and generate vascular smooth muscle cells (VSMCs). Risk VSMCs exhibit globally altered transcriptional networks that intersect with previously identified CAD risk genes and pathways, concomitant with aberrant adhesion, contraction, and proliferation. Unexpectedly, deleting the risk haplotype rescues VSMC stability, while expressing the 9p21.3-associated long non-coding RNA ANRIL induces risk phenotypes in non-risk VSMCs. This study shows that the risk haplotype selectively predisposes VSMCs to adopt a cell state associated with CAD phenotypes, defines new VSMC-based networks of CAD risk genes, and establishes haplotype-edited iPSCs as powerful tools for functionally annotating the human genome.
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•Profiling iPSC-derived VSMCs uncovers CAD risk haplotype-dependent phenotypes•Deleting the risk haplotype rescues VSMC proliferation, adhesion, and contraction•Risk-dependent gene networks drive cell state instability, partially through ANRIL•Evidence for cross-talk between CAD risk loci predicts vascular therapeutic targets
Genome editing and iPSC-based modeling of the 9p21.3 cardiovascular disease locus shows that it controls large gene networks acting in vascular smooth muscle cells that predispose them to assume atherogenic-like cell states.
Small molecules targeting allosteric pockets of G protein-coupled receptors (GPCRs) have a great therapeutic potential for the treatment of neurologic and other chronic disorders. Here we performed ...virtual screening for orthosteric and putative allosteric ligands of the human dopamine D3 receptor (D3R) using two optimized crystal-structure-based models: the receptor with an empty binding pocket (D3R(APO)), and the receptor complex with dopamine (D3R(Dopa)). Subsequent biochemical and functional characterization revealed 14 novel ligands with a binding affinity of better than 10 μM in the D3R(APO) candidate list (56% hit rate), and 8 novel ligands in the D3R(Dopa) list (32% hit rate). Most ligands in the D3R(APO) model span both orthosteric and extended pockets and behave as antagonists at D3R, with compound 7 showing the highest potency of dopamine inhibition (IC₅₀ = 7 nM). In contrast, compounds identified by the D3R(Dopa) model are predicted to occupy an allosteric site at the extracellular extension of the pocket, and they all lack the anchoring amino group. Compounds targeting the allosteric site display a variety of functional activity profiles, where behavior of at least two compounds (23 and 26) is consistent with noncompetitive allosteric modulation of dopamine signaling in the extracellular signal-regulated kinase 1 and 2 phosphorylation and β-arrestin recruitment assays. The high affinity and ligand efficiency of the chemically diverse hits identified in this study suggest utility of structure-based screening targeting allosteric sites of GPCRs.
The transcriptional programs that establish neuronal identity evolved to produce the rich diversity of neuronal cell types that arise sequentially during development. Remarkably, transient expression ...of certain transcription factors can also endow non-neural cells with neuronal properties. The relationship between reprogramming factors and the transcriptional networks that produce neuronal identity and diversity remains largely unknown. Here, from a screen of 598 pairs of transcription factors, we identify 76 pairs of transcription factors that induce mouse fibroblasts to differentiate into cells with neuronal features. By comparing the transcriptomes of these induced neuronal cells (iN cells) with those of endogenous neurons, we define a 'core' cell-autonomous neuronal signature. The iN cells also exhibit diversity; each transcription factor pair produces iN cells with unique transcriptional patterns that can predict their pharmacological responses. By linking distinct transcription factor input 'codes' to defined transcriptional outputs, this study delineates cell-autonomous features of neuronal identity and diversity and expands the reprogramming toolbox to facilitate engineering of induced neurons with desired patterns of gene expression and related functional properties.
Somatic mutation in neurons is linked to neurologic disease and implicated in cell-type diversification. However, the origin, extent, and patterns of genomic mutation in neurons remain unknown. We ...established a nuclear transfer method to clonally amplify the genomes of neurons from adult mice for whole-genome sequencing. Comprehensive mutation detection and independent validation revealed that individual neurons harbor ∼100 unique mutations from all classes but lack recurrent rearrangements. Most neurons contain at least one gene-disrupting mutation and rare (0–2) mobile element insertions. The frequency and gene bias of neuronal mutations differ from other lineages, potentially due to novel mechanisms governing postmitotic mutation. Fertile mice were cloned from several neurons, establishing the compatibility of mutated adult neuronal genomes with reprogramming to pluripotency and development.
•Reprogramming neurons by cloning enables high-fidelity whole-genome sequencing•Neurons harbor ∼100 unique mutations but lack recurrent DNA rearrangements•Neuronal mutations impact expressed genes and exhibit unique molecular signatures•Mature adult neurons can generate fertile adult mouse clones
Hazen et al. use cloning to amplify and perform complete genome sequence analyses on adult neurons. They discover unique characteristics of neuronal genomes consistent with postmitotic mutation and further establish neuronal genomic integrity by generating fertile mice from these neurons.
Cardiovascular disease (CVD) is the most common cause of death in the world to date. Yet, our understanding of molecular mechanisms, as well as environmental and genetic predisposition is limited. ...The 9p21.3 CVD risk locus has the largest genetic impact within the population, however the polymorphisms are present only in DNA regulatory elements, and not in the exons, the resident long non-coding RNA (ANRIL), nor the protein coding genes (CDKN2A/B). We used patient specific iPSCs and genome editing in our isogenic approach to understand the function of the locus in cultured vascular smooth muscle cells (VSMCs). Interestingly, we observed a change in morphology and function of the risk allele homozygous SMCs, associated with overexpressed short ANRIL isoform, which upon whole transcriptome examination seemed to resemble cell identity switch or dedifferentiation. Remarkably, the pathway analysis on the differentially expressed genes revealed unification of known CVD GWAS loci and regulatory molecules (mTOR, PPAR, PDGF, TGFβ, PTGER2, etc.) involved in CVD within our single gene model. In addition, we observed perturbations in pathways previously unconnected to CVD. Single cell RNA-seq analysis of ANRIL overexpression in SMCs suggests at least two stage non-deterministic identity transition from contractile SMCs to proliferative phenotype and recapitulates many known and new pathways found in the bulk transcriptome analysis.
Pharmacological responses of G protein-coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR ...structures. We re-engineered the human A
2A
adenosine receptor by replacing its third intracellular loop with apo-cytochrome b562RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure identified about 57 ordered waters inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved Asp
2.50
. Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured waters, sodium ions and lipids/cholesterol in GPCR stabilization and function.
We apply the spin-fermion model to study the normal state and pairing
instability in electron-doped cuprates near the antiferromagnetic QCP. Peculiar
frequency dependencies of the normal state ...properties are shown to emerge from
the self-consistent equations on the fermionic and bosonic self-energies, and
are in agreement with experimentally observed ones. We argue that the pairing
instability is in the $d_{x^{2}-y^{2}}$ channel, as in hole-doped cuprates, but
theoretical $T_{c}$ is much lower than in the hole-doped case. For the same
hopping integrals and the interaction strength as in hole-doped materials, we
obtain $T_{c}\sim10$K at the end point of the antiferromagnetic phase. We argue
that a strong reduction of $T_{c}$ in electron-doped cuprates compared to
hole-doped ones is due to critical role of the Fermi surface curvature for
electron-doped materials. The $d_{x^{2}-y^{2}}$-pairing gap
$\Delta(\mathbf{k},\omega)$ is strongly non-monotonic along the Fermi surface.
The position of the gap maxima, however, does not coincide with the hot spots,
as the non-monotonic $d_{x^{2}-y^{2}}$ gap persists even at doping when the hot
spots merge on the Brillouin zone diagonals.
Phys. Rev. Lett. 96, 107002 (2006) We study the normal state and pairing instability in electron-doped cuprates
near optimal doping. We show that the fermionic self-energy has a non-Fermi
liquid form ...leading to peculiar frequency dependencies of the conductivity and
the Raman response. We solve the pairing problem and demonstrate that T_c is
determined by the curvature of the Fermi surface, and the pairing gap
\Delta(k,\omega) is strongly non-monotonic along the Fermi surface. The normal
state frequency dependencies, the value of T_c\sim10K and the k-dependence of
the gap agree with the experiment.
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