Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation ...predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.
The psychiatric disorders autism and schizophrenia have a strong genetic component, and copy number variants (CNVs) are firmly implicated. Recurrent deletions and duplications of chromosome 16p11.2 ...confer a high risk for both diseases, but the pathways disrupted by this CNV are poorly defined. Here we investigate the dynamics of the 16p11.2 network by integrating physical interactions of 16p11.2 proteins with spatiotemporal gene expression from the developing human brain. We observe profound changes in protein interaction networks throughout different stages of brain development and/or in different brain regions. We identify the late mid-fetal period of cortical development as most critical for establishing the connectivity of 16p11.2 proteins with their co-expressed partners. Furthermore, our results suggest that the regulation of the KCTD13-Cul3-RhoA pathway in layer 4 of the inner cortical plate is crucial for controlling brain size and connectivity and that its dysregulation by de novo mutations may be a potential determinant of 16p11.2 CNV deletion and duplication phenotypes.
•Rare high-risk CNVs for psychiatric disorders have unique spatiotemporal signatures•Dynamic 16p11.2 protein interaction network reveals changes during brain development•The late mid-fetal period is critical for establishing 16p11.2 network connectivity•KCTD13-Cul3-RhoA pathway may be dysregulated by gene-damaging mutations
Deletions and duplications of chromosome 16p11.2 confer a high risk for neuropsychiatric diseases. By integrating the physical interactions of 16p11.2 proteins with spatiotemporal gene expression, Lin et al. implicate the KCTD13-Cul3-RhoA pathway as being crucial for controlling brain size and connectivity.
During the course of a viral infection, viral proteins interact with an array of host proteins and pathways. Here, we present a systematic strategy to elucidate the dynamic interactions between H1N1 ...influenza and its human host. A combination of yeast two-hybrid analysis and genome-wide expression profiling implicated hundreds of human factors in mediating viral-host interactions. These factors were then examined functionally through depletion analyses in primary lung cells. The resulting data point to potential roles for some unanticipated host and viral proteins in viral infection and the host response, including a network of RNA-binding proteins, components of WNT signaling, and viral polymerase subunits. This multilayered approach provides a comprehensive and unbiased physical and regulatory model of influenza-host interactions and demonstrates a general strategy for uncovering complex host-pathogen relationships.
A major cause of enteric infection, Gram-negative pathogenic bacteria activate mucosal inflammation through lipopolysaccharide (LPS) binding to intestinal toll-like receptor 4 (TLR4). Breast feeding ...lowers risk of disease, and human milk modulates inflammation.
This study tested whether human milk oligosaccharides (HMOSs) influence pathogenic Escherichia coli-induced interleukin (IL)-8 release by intestinal epithelial cells (IECs), identified specific proinflammatory signalling molecules modulated by HMOSs, specified the active HMOS and determined its mechanism of action.
Models of inflammation were IECs invaded by type 1 pili enterotoxigenic E. coli (ETEC) in vitro: T84 modelled mature, and H4 modelled immature IECs. LPS-induced signalling molecules co-varying with IL-8 release in the presence or absence of HMOSs were identified. Knockdown and overexpression verified signalling mediators. The oligosaccharide responsible for altered signalling was identified.
HMOSs attenuated LPS-dependent induction of IL-8 caused by ETEC, uropathogenic E. coli, and adherent-invasive E. coli (AIEC) infection, and suppressed CD14 transcription and translation. CD14 knockdown recapitulated HMOS-induced attenuation. Overexpression of CD14 increased the inflammatory response to ETEC and sensitivity to inhibition by HMOSs. 2'-fucosyllactose (2'-FL), at milk concentrations, displayed equivalent ability as total HMOSs to suppress CD14 expression, and protected AIEC-infected mice.
HMOSs and 2'-FL directly inhibit LPS-mediated inflammation during ETEC invasion of T84 and H4 IECs through attenuation of CD14 induction. CD14 expression mediates LPS-TLR4 stimulation of portions of the 'macrophage migration inhibitory factors' inflammatory pathway via suppressors of cytokine signalling 2/signal transducer and activator of transcription 3/NF-κB. HMOS direct inhibition of inflammation supports its functioning as an innate immune system whereby the mother protects her vulnerable neonate through her milk. 2'-FL, a principal HMOS, quenches inflammatory signalling.
Recent advances in DNA/RNA sequencing have made it possible to identify new targets rapidly and to repurpose approved drugs for treating heterogeneous diseases by the 'precise' targeting of ...individualized disease modules. In this study, we develop a Genome-wide Positioning Systems network (GPSnet) algorithm for drug repurposing by specifically targeting disease modules derived from individual patient's DNA and RNA sequencing profiles mapped to the human protein-protein interactome network. We investigate whole-exome sequencing and transcriptome profiles from ~5,000 patients across 15 cancer types from The Cancer Genome Atlas. We show that GPSnet-predicted disease modules can predict drug responses and prioritize new indications for 140 approved drugs. Importantly, we experimentally validate that an approved cardiac arrhythmia and heart failure drug, ouabain, shows potential antitumor activities in lung adenocarcinoma by uniquely targeting a HIF1α/LEO1-mediated cell metabolism pathway. In summary, GPSnet offers a network-based, in silico drug repurposing framework for more efficacious therapeutic selections.
One of the most oft-employed methods for C–C bond formation involving the coupling of vinyl-halides with aldehydes catalyzed by Ni and Cr (Nozaki–Hiyama–Kishi, NHK) has been rendered more practical ...using an electroreductive manifold. Although early studies pointed to the feasibility of such a process, those precedents were never applied by others due to cumbersome setups and limited scope. Here we show that a carefully optimized electroreductive procedure can enable a more sustainable approach to NHK, even in an asymmetric fashion on highly complex medicinally relevant systems. The e-NHK can even enable non-canonical substrate classes, such as redox-active esters, to participate with low loadings of Cr when conventional chemical techniques fail. A combination of detailed kinetics, cyclic voltammetry, and in situ UV–vis spectroelectrochemistry of these processes illuminates the subtle features of this mechanistically intricate process.
The study and application of transition metal hydrides (TMHs) has been an active area of chemical research since the early 1960s
, for energy storage, through the reduction of protons to generate ...hydrogen
, and for organic synthesis, for the functionalization of unsaturated C-C, C-O and C-N bonds
. In the former instance, electrochemical means for driving such reactivity has been common place since the 1950s
but the use of stoichiometric exogenous organic- and metal-based reductants to harness the power of TMHs in synthetic chemistry remains the norm. In particular, cobalt-based TMHs have found widespread use for the derivatization of olefins and alkynes in complex molecule construction, often by a net hydrogen atom transfer (HAT)
. Here we show how an electrocatalytic approach inspired by decades of energy storage research can be made use of in the context of modern organic synthesis. This strategy not only offers benefits in terms of sustainability and efficiency but also enables enhanced chemoselectivity and distinct, tunable reactivity. Ten different reaction manifolds across dozens of substrates are exemplified, along with detailed mechanistic insights into this scalable electrochemical entry into Co-H generation that takes place through a low-valent intermediate.
Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D'Arcy Hart showed that Mtb avoids delivery to lysosomes, but ...the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown. Here, we describe a systematic strategy to identify bacterial virulence factors by looking for interactions between the Mtb secretome and host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform. Using this approach we identified an interaction between EsxH, which is secreted by the Esx-3 TSSS, and human hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). ESCRT has a well-described role in directing proteins destined for lysosomal degradation into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), ensuring degradation of the sorted cargo upon MVB-lysosome fusion. Here, we show that ESCRT is required to deliver Mtb to the lysosome and to restrict intracellular bacterial growth. Further, EsxH, in complex with EsxG, disrupts ESCRT function and impairs phagosome maturation. Thus, we demonstrate a role for a TSSS and the host ESCRT machinery in one of the central features of tuberculosis pathogenesis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
GPCRs modulate a plethora of physiological processes and mediate the effects of one‐third of FDA‐approved drugs. Depending on which ligand activates a receptor, it can engage different intracellular ...transducers. This ‘biased signalling’ paradigm requires that we now characterize physiological signalling not just by receptors but by ligand–receptor pairs. Ligands eliciting biased signalling may constitute better drugs with higher efficacy and fewer adverse effects. However, ligand bias is very complex, making reproducibility and description challenging. Here, we provide guidelines and terminology for any scientists to design and report ligand bias experiments. The guidelines will aid consistency and clarity, as the basic receptor research and drug discovery communities continue to advance our understanding and exploitation of ligand bias. Scientific insight, biosensors, and analytical methods are still evolving and should benefit from and contribute to the implementation of the guidelines, together improving translation from in vitro to disease‐relevant in vivo models.
The central circadian clock within the suprachiasmatic nucleus (SCN) plays an important role in temporally organizing and coordinating many of the processes governing cancer cell proliferation and ...tumor growth in synchrony with the daily light/dark cycle which may contribute to endogenous cancer prevention. Bioenergetic substrates and molecular intermediates required for building tumor biomass each day are derived from both aerobic glycolysis (Warburg effect) and lipid metabolism. Using tissue-isolated human breast cancer xenografts grown in nude rats, we determined that circulating systemic factors in the host and the Warburg effect, linoleic acid uptake/metabolism and growth signaling activities in the tumor are dynamically regulated, coordinated and integrated within circadian time structure over a 24-hour light/dark cycle by SCN-driven nocturnal pineal production of the anticancer hormone melatonin. Dim light at night (LAN)-induced melatonin suppression disrupts this circadian-regulated host/cancer balance among several important cancer preventative signaling mechanisms, leading to hyperglycemia and hyperinsulinemia in the host and runaway aerobic glycolysis, lipid signaling and proliferative activity in the tumor.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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