Discovery of Protein-Protein Interaction Inhibitors by Integrating Protein Engineering and Chemical Screening Platforms

Protein-protein interactions (PPIs) govern intracellular life, and identification of PPI inhibitors is challenging. Roadblocks in assay development stemming from weak binding affinities of natural PPIs impede progress in this field. We postulated that enhancing binding affinity of natural PPIs via protein engineering will aid assay development and hit discovery. This proof-of-principle study targets PPI between linear ubiquitin chains and NEMO UBAN domain, which activates NF-κB signaling. Using phage display, we generated ubiquitin variants that bind to the functional UBAN epitope with high affinity, act as competitive inhibitors, and structurally maintain the existing PPI interface. When utilized in assay development, variants enable generation of robust cell-based assays for chemical screening. Top compounds identified using this approach directly bind to UBAN and dampen NF-κB signaling. This study illustrates advantages of integrating protein engineering and chemical screening in hit identification, a development that we anticipate will have wide application in drug discovery

A Disk Around the Planetary-Mass Companion GSC 06214-00210 b: Clues About the Formation of Gas Giants on Wide Orbits

We present Keck/OSIRIS 1.1-1.8 um adaptive optics integral field spectroscopy of the planetary-mass companion to GSC 06214-00210, a member of the ~5 Myr Upper Scorpius OB association. We infer a spectral type of L0+/-1, and our spectrum exhibits multiple signs of youth. The most notable feature is exceptionally strong PaBeta emission (EW=-11.4 +/- 0.3 A) which signals the presence of a circumplanetary accretion disk. The luminosity of GSC 06214-00210 b combined with its age yields a model-dependent mass of 14 +/- 2 MJup, making it the lowest-mass companion to show evidence of a disk. With a projected separation of 320 AU, the formation of GSC 06214-00210 b and other very low-mass companions on similarly wide orbits is unclear. One proposed mechanism is formation at close separations followed by planet-planet scattering to much larger orbits. Since that scenario involves a close encounter with another massive body, which is probably destructive to circumplanetary disks, it is unlikely that GSC 06214-00210 b underwent a scattering event in the past. This implies that planet-planet scattering is not solely responsible for the population of gas giants on wide orbits. More generally, the identification of disks around young planetary companions on wide orbits offers a novel method to constrain the formation pathway of these objects, which is otherwise notoriously difficult to do for individual systems. We also refine the spectral type of the primary from M1 to K7 and detect a mild (2-sigma) excess at 22 um using WISE photometry.Comment: 25 pages, 13 figures; Accepted by Ap

The Runts of the Litter: Why planets formed through gravitational instability can only be failed binary stars

Recent direct imaging discoveries suggest a new class of massive, distant planets around A stars. These widely separated giants have been interpreted as signs of planet formation driven by gravitational instability, but the viability of this mechanism is not clear cut. In this paper, we first discuss the local requirements for fragmentation and the initial fragment mass scales. We then consider whether the fragment's subsequent growth can be terminated within the planetary mass regime. Finally, we place disks in the larger context of star formation and disk evolution models. We find that in order for gravitational instability to produce planets, disks must be atypically cold in order to reduce the initial fragment mass. In addition, fragmentation must occur during a narrow window of disk evolution, after infall has mostly ceased, but while the disk is still sufficiently massive to undergo gravitational instability. Under more typical conditions, disk-born objects will likely grow well above the deuterium burning planetary mass limit. We conclude that if planets are formed by gravitational instability, they must be the low mass tail of the distribution of disk-born companions. To validate this theory, on-going direct imaging surveys must find a greater abundance of brown dwarf and M-star companions to A-stars. Their absence would suggest planet formation by a different mechanism such as core accretion, which is consistent with the debris disks detected in these systems.Comment: 14 pages, 6 figures, accepted to ApJ, some content changes, revised figure

General Anesthetics Predicted to Block the GLIC Pore with Micromolar Affinity

Although general anesthetics are known to modulate the activity of ligand-gated ion channels in the Cys-loop superfamily, there is at present neither consensus on the underlying mechanisms, nor predictive models of this modulation. Viable models need to offer quantitative assessment of the relative importance of several identified anesthetic binding sites. However, to date, precise affinity data for individual sites has been challenging to obtain by biophysical means. Here, the likely role of pore block inhibition by the general anesthetics isoflurane and propofol of the prokaryotic pentameric channel GLIC is investigated by molecular simulations. Microscopic affinities are calculated for both single and double occupancy binding of isoflurane and propofol to the GLIC pore. Computations are carried out for an open-pore conformation in which the pore is restrained to crystallographic radius, and a closed-pore conformation that results from unrestrained molecular dynamics equilibration of the structure. The GLIC pore is predicted to be blocked at the micromolar concentrations for which inhibition by isofluorane and propofol is observed experimentally. Calculated affinities suggest that pore block by propofol occurs at signifcantly lower concentrations than those for which inhibition is observed: we argue that this discrepancy may result from binding of propofol to an allosteric site recently identified by X-ray crystallography, which may cause a competing gain-of-function effect. Affinities of isoflurane and propofol to the allosteric site are also calculated, and shown to be 3 mM for isoflurane and for propofol; both anesthetics have a lower affinity for the allosteric site than for the unoccupied pore

P A ON THE DIVISORS OF ORDER r

Abstract: N. Minculete has introduced the divisor-of-order-r function τ (r) and the sum-of-divisors-of-order-r function σ (r) . We investigate the asymptotic behaviour of n≤x τ (r) (n) and n≤x σ (r) (n) and improve known estimates

Disrupting the LC3 interaction region (LIR) binding of selective autophagy receptors sensitizes AML cell lines to cytarabine

Short linear motifs (SLiMs) located in disordered regions of multidomain proteins are important for the organization of protein–protein interaction networks. By dynamic association with their binding partners, SLiMs enable assembly of multiprotein complexes, pivotal for the regulation of various aspects of cell biology in higher organisms. Despite their importance, there is a paucity of molecular tools to study SLiMs of endogenous proteins in live cells. LC3 interacting regions (LIRs), being quintessential for orchestrating diverse stages of autophagy, are a prominent example of SLiMs and mediate binding to the ubiquitin-like LC3/GABARAP family of proteins. The role of LIRs ranges from the posttranslational processing of their binding partners at early stages of autophagy to the binding of selective autophagy receptors (SARs) to the autophagosome. In order to generate tools to study LIRs in cells, we engineered high affinity binders of LIR motifs of three archetypical SARs: OPTN, p62, and NDP52. In an array of in vitro and cellular assays, the engineered binders were shown to have greatly improved affinity and specificity when compared with the endogenous LC3/GABARAP family of proteins, thus providing a unique possibility for modulating LIR interactions in living systems. We exploited these novel tools to study the impact of LIR inhibition on the fitness and the responsiveness to cytarabine treatment of THP-1 cells – a model for studying acute myeloid leukemia (AML). Our results demonstrate that inhibition of LIR of a single autophagy receptor is insufficient to sensitize the cells to cytarabine, while simultaneous inhibition of three LIR motifs in three distinct SARs reduces the IC50 of the chemotherapeutic

Effect of External Factors on Trace Element Profile and Biomass of Mustard (Brássica júncea L.) Microgreens: Neural Network Analysis

Growing organic microgreens indoors requires a unified technological procedure with various external elicitors. The quality of seedlings depends on their ability to accumulate essential microelements. This research assessed the nutrient profile of mustard microgreens using the method of fractal calculation with repeating numerical series. The experiment involved mustard (Brássica júncea L.) of the Nika variety grown in a closed box for 15 days under aggregation with an intensive 16-h photocycle (440 µmoL m2/s). The plants were inoculated with the endomycorrhizal fungus Glomus mos- seae. A solution of fulvic acids (100 mg/L) served as a stabilizing organic additive and was introduced into the coconut substrate. The physical treatment included weak static electromagnetic field with magnetic induction (20 mT). The elemental analysis was performed by inductively coupled plasma atomic emission spectrometry on an ICPE-9000 device (Shimadzu, Japan). According to the calculated indices of the microelement biocomposition, the best result belonged to the sample treated with fulvic acids and weak electromagnetic field (IndBcomL = 0.27). The resulting biomass of dry powder for elemental analysis was 10.2 g, which was twice as high as the values obtained in the control sample, not subjected to any external influences (5.2 g). All the variants with mycorrhization produced no positive effect on the total pool of microelements during vegetation. The increase in biomass averaged as low as 20%. Zinc increased by 33.3% while aluminum and iron decreased by 59.5 and 18.0%, respectively. The neural network analysis of the microelements in mustard microgreens proved effective as a mathematical model for biochemical diagnostics of biomass quality. The method could be used to optimize the biotechnological process for other indoor crops as it makes it possible to partially substitute mineral fertilizers with organic and bacterial complex

A demonstration of modularity, reuse, reproducibility, portability and scalability for modeling and simulation of cardiac electrophysiology using Kepler Workflows.

Multi-scale computational modeling is a major branch of computational biology as evidenced by the US federal interagency Multi-Scale Modeling Consortium and major international projects. It invariably involves specific and detailed sequences of data analysis and simulation, often with multiple tools and datasets, and the community recognizes improved modularity, reuse, reproducibility, portability and scalability as critical unmet needs in this area. Scientific workflows are a well-recognized strategy for addressing these needs in scientific computing. While there are good examples if the use of scientific workflows in bioinformatics, medical informatics, biomedical imaging and data analysis, there are fewer examples in multi-scale computational modeling in general and cardiac electrophysiology in particular. Cardiac electrophysiology simulation is a mature area of multi-scale computational biology that serves as an excellent use case for developing and testing new scientific workflows. In this article, we develop, describe and test a computational workflow that serves as a proof of concept of a platform for the robust integration and implementation of a reusable and reproducible multi-scale cardiac cell and tissue model that is expandable, modular and portable. The workflow described leverages Python and Kepler-Python actor for plotting and pre/post-processing. During all stages of the workflow design, we rely on freely available open-source tools, to make our workflow freely usable by scientists