Rescue of oxytocin response and social behaviour in a mouse model of autism

A fundamental challenge in developing treatments for autism spectrum disorders is the heterogeneity of the condition. More than one hundred genetic mutations confer high risk for autism, with each individual mutation accounting for only a small fraction of cases1,2,3. Subsets of risk genes can be grouped into functionally related pathways, most prominently those involving synaptic proteins, translational regulation, and chromatin modifications. To attempt to minimize this genetic complexity, recent therapeutic strategies have focused on the neuropeptides oxytocin and vasopressin4,5,6, which regulate aspects of social behaviour in mammals7. However, it is unclear whether genetic risk factors predispose individuals to autism as a result of modifications to oxytocinergic signalling. Here we report that an autism-associated mutation in the synaptic adhesion molecule Nlgn3 results in impaired oxytocin signalling in dopaminergic neurons and in altered behavioural responses to social novelty tests in mice. Notably, loss of Nlgn3 is accompanied by a disruption of translation homeostasis in the ventral tegmental area. Treatment of Nlgn3-knockout mice with a new, highly specific, brain-penetrant inhibitor of MAP kinase-interacting kinases resets the translation of mRNA and restores oxytocin signalling and social novelty responses. Thus, this work identifies a convergence between the genetic autism risk factor Nlgn3, regulation of translation, and oxytocinergic signalling. Focusing on such common core plasticity elements might provide a pragmatic approach to overcoming the heterogeneity of autism. Ultimately, this would enable mechanism-based stratification of patient populations to increase the success of therapeutic interventions

Topography of transcriptionally active chromatin in glioblastoma

Molecular profiling of the most aggressive brain tumor glioblastoma (GBM) on the basis of gene expression, DNA methylation, and genomic variations advances both cancer research and clinical diagnosis. The enhancer architectures and regulatory circuitries governing tumor-intrinsic transcriptional diversity and subtype identity are still elusive. Here, by mapping H3K27ac deposition, we analyze the active regulatory landscapes across 95 GBM biopsies, 12 normal brain tissues, and 38 cell line counterparts. Analyses of differentially regulated enhancers and super-enhancers uncovered previously unrecognized layers of intertumor heterogeneity. Integrative analysis of variant enhancer loci and transcriptome identified topographies of transcriptional enhancers and core regulatory circuitries in four molecular subtypes of primary tumors: AC1-mesenchymal, AC1-classical, AC2-proneural, and AC3-proneural. Moreover, this study reveals core oncogenic dependency on super-enhancer–driven transcriptional factors, long noncoding RNAs, and druggable targets in GBM. Through profiling of transcriptional enhancers, we provide clinically relevant insights into molecular classification, pathogenesis, and therapeutic intervention of GBM

MNK1 and MNK2 enforce expression of E2F1, FOXM1, and WEE1 to drive soft tissue sarcoma

Soft tissue sarcoma (STS) is a heterogeneous disease that arises from connective tissues. Clinical outcome of patients with advanced tumors especially de-differentiated liposarcoma and uterine leiomyosarcoma remains unsatisfactory, despite intensive treatment regimens including maximal surgical resection, radiation, and chemotherapy. MAP kinase-interacting serine/threonine-protein kinase 1 and 2 (MNK1/2) have been shown to contribute to oncogenic translation via phosphorylation of eukaryotic translation initiation factor 4E (eIF4E). However, little is known about the role of MNK1/2 and their downstream targets in STS. In this study, we show that depletion of either MNK1 or MNK2 suppresses cell viability, anchorage-independent growth, and tumorigenicity of STS cells. We also identify a compelling antiproliferative efficacy of a novel, selective MNK inhibitor ETC-168. Cellular responsiveness of STS cells to ETC-168 correlates positively with that of phosphorylated ribosomal protein S6 (RPS6). Mirroring MNK1/2 silencing, ETC-168 treatment strongly blocks eIF4E phosphorylation and represses expression of sarcoma-driving onco-proteins including E2F1, FOXM1, and WEE1. Moreover, combination of ETC-168 and MCL1 inhibitor S63845 exerts a synergistic antiproliferative activity against STS cells. In summary, our study reveals crucial roles of MNK1/2 and their downstream targets in STS tumorigenesis. Our data encourage further clinical translation of MNK inhibitors for STS treatment

Effect of Ser392 Phosphorylation on the Structure and Dynamics of the Polybasic Domain of ADP Ribosylation Factor Nucleotide Site Opener Protein: A Molecular Simulation Study

ADP ribosylation factor nucleotide site opener (ARNO) as a guanine nucleotide exchange factor (GEF) activates small GTPases called ADP ribosylation factors (Arfs), which function as molecular switches and regulate a variety of cell biological events. ARNO directly interacts with the transmembrane a2-subunit isoform of the proton-pumping vacuolar ATPase in an acidification-dependent manner, and this interaction plays a crucial role in the regulation of the protein degradation pathway. A recent study reported specific interactions of a2N with the ARNO_375–400 peptide corresponding to the polybasic (PB) domain of ARNO, which is a crucial regulatory element in the autoregulation and modulation of Arf-GEF activity. Interestingly, phosphorylation of Ser392 completely abolishes this interaction, and the experimental structure shows significant structural rearrangements. To investigate the effect of Ser392 phosphorylation on the structure and dynamics of the ARNO_375–400 peptide, we employed all atom molecular dynamics (MD) simulations of the phosphorylated and unphosphorylated PB domain of the ARNO protein. A Hamiltonian-based replica exchange method called biasing potential replica exchange MD was used to enhance conformational sampling. Simulations predicted that the isolated PB domain is highly flexible, with the C-terminal region of the unphosphorylated state being unstable. In contrast, Ser392 phosphorylation increases the overall stability of the peptide. In agreement with experimental results, our simulations further support the hypothesis that phosphorylation induces disorder to order transitions and provide new insights into the structural dynamics of the PB domain. Phosphorylation of Ser392 appears to stabilize the C-terminal α-helix via formation of salt bridges between phospho-Ser392 and Arg390, Lys395, and Lys396

β2-Chimaerin is a novel target for diacylglycerol: Binding properties and changes in subcellular localization mediated by ligand binding to its C1 domain

The members of the chimaerin family of Rac-GTPase-activating proteins possess a single C1 domain with high homology to those present in protein kinase C (PKC) isozymes. This domain in PKCs is involved in phorbol ester and diacylglycerol (DAG) binding. We previously have demonstrated that one of the chimaerin isoforms, β2-chimaerin, binds phorbol esters with high affinity. In this study we analyzed the properties of β2-chimaerin as a DAG receptor by using a series of conformationally constrained cyclic DAG analogues (DAG lactones) as probes. We identified analogs that bind to β2-chimaerin with more than 100-fold higher affinity than 1-oleoyl-2-acetylglycerol. The potencies of these analogs approach those of the potent phorbol ester tumor promoters. The different DAG lactones show some selectivity for this novel receptor compared with PKCα. Cellular studies revealed that these DAG analogs induce translocation of β2-chimaerin from cytosolic (soluble) to particulate fractions. Using green fluorescent protein-fusion proteins for β2-chimaerin we determined that this novel receptor translocates to the perinuclear region after treatment with DAG lactones. Binding and translocation were prevented by mutation of the conserved Cys-246 in the C1 domain. The structural homology between the C1 domain of β2-chimaerin and the C1b domain of PKCδ also was confirmed by modeling analysis. Our results demonstrate that β2-chimaerin is a high affinity receptor for DAG through binding to its C1 domain and supports the emerging concept that multiple pathways transduce signaling through DAG and the phorbol esters

Structural insights into the inhibition of Zika virus NS2B-NS3 protease by a small-molecule inhibitor

Zika virus (ZIKV) infection has become a global public health concern. The viral NS2B-NS3 protease is an attractive antiviral target because of its role in maturation of viral non-structural proteins. Substrate-derived protease inhibitors have been investigated, but it remains challenging to develop them into drugs. Small-molecule inhibitors are of great interest in antiviral drug development. Here we report the structure and dynamics of ZIKV NS2B-NS3 protease covalently bound to a small-molecule inhibitor. Our crystallographic and NMR studies demonstrate that the inhibitor further stabilizes the closed conformation of ZIKV protease. Upon hydrolysis in situ into two fragments, the benzoyl group of the inhibitor forms a covalent bond with the side chain of catalytic residue S135, whereas the second fragment exhibits no obvious molecular interactions with the protease. This study provides a detailed mechanism of action for a covalent inhibitor, which will guide further development of ZIKV protease inhibitors

Probing the binding mechanism of Mnk inhibitors by docking and molecular dynamics simulations

10.1021/bi501261jBiochemistry54132-4