Peptides that contain β-amino acids display stable secondary structures, such as helices and sheets, and are often referred to as foldamers. Cyclic β
-amino acids (cβAAs), such as ...2-aminocyclohexanecarboxylic acid (2-ACHC), are strong helix/turn inducers due to their restricted conformations. Here we report the ribosomal synthesis of foldamer peptides that contain multiple, up to ten, consecutive cβAAs via genetic code reprogramming. We also report the de novo discovery of macrocyclic cβAA-containing peptides capable of binding to a protein target. As a demonstration, potent binders with low-to-subnanomolar K
values were identified for human factor XIIa (hFXIIa) and interferon-gamma receptor 1, from a library of their 10
members. One of the anti-hFXIIa macrocyclic peptides that exhibited a high inhibitory activity and serum stability was co-crystallized with hFXIIa. The X-ray structure revealed that it adopts an antiparallel β-sheet structure induced by a (1S,2S)-2-ACHC residue via the formation of two γ-turns. This work demonstrates the potential of this platform to explore the previously inaccessible sequence space of cβAA-containing peptides.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The mutual exclusion protocol invented by Mellor-Crummey and Scott (called MCS protocol) is used to exemplify that state picture designs based on which the state machine graphical animation (SMGA) ...tool produces graphical animations should be better visualized. Variants of MCS protocol have been used in Java virtual machines and therefore the 2006 Edsger W. Dijkstra Prize in Distributed Computing went to their paper on MCS protocol. The new state picture design of a state machine formalizing MCS protocol is assessed based on Gestalt principles, more specifically proximity principle and similarity principle. We report on a core part of a formal verification case study in which the new state picture design and the SMGA tool largely contributed to the successful completion of the formal proof that MCS protocol enjoys the mutual exclusion property. The lessons learned acquired through our experiments are summarized as two groups of tips. The first group is some new tips on how to make state picture designs. The second one is some tips on how to conjecture state machine characteristics by using the SMGA tool. We also report on one more case study in which the state picture design has been made for the mutual exclusion protocol invented by Anderson (called Anderson protocol) and some characteristics of the protocol have been discovered based on the tips.
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CEKLJ, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Dimethylated histone H3 Lys36 (H3K36me2) regulates gene expression, and aberrant H3K36me2 upregulation, resulting from either the overexpression or point mutation of the dimethyltransferase NSD2, is ...found in various cancers. Here we report the cryo-electron microscopy structure of NSD2 bound to the nucleosome. Nucleosomal DNA is partially unwrapped, facilitating NSD2 access to H3K36. NSD2 interacts with DNA and H2A along with H3. The NSD2 autoinhibitory loop changes its conformation upon nucleosome binding to accommodate H3 in its substrate-binding cleft. Kinetic analysis revealed that two oncogenic mutations, E1099K and T1150A, increase NSD2 catalytic turnover. Molecular dynamics simulations suggested that in both mutants, the autoinhibitory loop adopts an open state that can accommodate H3 more often than the wild-type. We propose that E1099K and T1150A destabilize the interactions that keep the autoinhibitory loop closed, thereby enhancing catalytic turnover. Our analyses guide the development of specific inhibitors of NSD2.
Abstract
Multi-modal interactions are frequently observed in intrinsically disordered regions (IDRs) of proteins upon binding to their partners. In many cases, post-translational modifications in ...IDRs are accompanied by coupled folding and binding. From both molecular simulations and biochemical experiments with mutational studies, we show that the IDR including a Ser rich region (SRR) of the transcription factor Ets1, just before the DNA-binding core domain, undergoes multi-modal interactions when the SRR is not phosphorylated. In the phosphorylated state, the SRR forms a few specific complex structures with the Ets1 core, covering the recognition helix in the core and drastically reducing the DNA binding affinities as the auto-inhibitory state. The binding kinetics of mutated Ets1 indicates that aromatic residues in the SRR can be substituted with other hydrophobic residues for the interactions with the Ets1 core.
Bioengineering of ribosomally synthesized and post-translationally modified peptides (RiPPs) is an emerging approach to explore the diversity of pseudo-natural product structures for drug discovery ...purposes. However, despite the initial advances in this area, bioactivity reprogramming of multienzyme RiPP biosynthetic pathways remains a major challenge. Here, we report a platform for de novo discovery of functional thiopeptides based on reengineered biosynthesis of lactazole A, a RiPP natural product assembled by five biosynthetic enzymes. The platform combines in vitro biosynthesis of lactazole-like thiopeptides and mRNA display to prepare and screen large (≥1012) combinatorial libraries of pseudo-natural products. We demonstrate the utility of the developed protocols in an affinity selection against Traf2- and NCK-interacting kinase (TNIK), a protein involved in several cancers, which yielded a plethora of candidate thiopeptides. Of the 11 synthesized compounds, 9 had high affinities for the target kinase (best K D = 1.2 nM) and 10 inhibited its enzymatic activity (best K i = 3 nM). X-ray structural analysis of the TNIK/thiopeptide interaction revealed the unique mode of substrate-competitive inhibition exhibited by two of the discovered compounds. The thiopeptides internalized to the cytosol of HEK293H cells as efficiently as the known cell-penetrating peptide Tat (4–6 μM). Accordingly, the most potent compound, TP15, inhibited TNIK in HCT116 cells. Altogether, our platform enables the exploration of pseudo-natural thiopeptides with favorable pharmacological properties in drug discovery applications.
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IJS, KILJ, NUK, PNG, UL, UM
Objective
Focal cortical dysplasia (FCD) type IIb is a cortical malformation characterized by cortical architectural abnormalities, dysmorphic neurons, and balloon cells. It has been suggested that ...FCDs are caused by somatic mutations in cells in the developing brain. Here, we explore the possible involvement of somatic mutations in FCD type IIb.
Methods
We collected a total of 24 blood‐brain paired samples with FCD, including 13 individuals with FCD type IIb, 5 with type IIa, and 6 with type I. We performed whole‐exome sequencing using paired samples from 9 of the FCD type IIb subjects. Somatic MTOR mutations were identified and further investigated using all 24 paired samples by deep sequencing of the entire gene's coding region. Somatic MTOR mutations were confirmed by droplet digital polymerase chain reaction. The effect of MTOR mutations on mammalian target of rapamycin (mTOR) kinase signaling was evaluated by immunohistochemistry and Western blotting analyses of brain samples and by in vitro transfection experiments.
Results
We identified four lesion‐specific somatic MTOR mutations in 6 of 13 (46%) individuals with FCD type IIb showing mutant allele rates of 1.11% to 9.31%. Functional analyses showed that phosphorylation of ribosomal protein S6 in FCD type IIb brain tissues with MTOR mutations was clearly elevated, compared to control samples. Transfection of any of the four MTOR mutants into HEK293T cells led to elevated phosphorylation of 4EBP, the direct target of mTOR kinase.
Interpretation
We found low‐prevalence somatic mutations in MTOR in FCD type IIb, indicating that activating somatic mutations in MTOR cause FCD type IIb. Ann Neurol 2015;78:375–386
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The paper proposes a new testing technique for concurrent programs. The technique is a specification-based testing. For a formal specification S and a concurrent program P , state sequences are ...generated from P and checked to be accepted by S . We suppose that S is specified in Maude and P is implemented in Java. Java Pathfinder (JPF) and Maude are then used to generate state sequences from P and to check if such state sequences are accepted by S , respectively. Even without checking any property violations with JPF, JPF often encounters the notorious state space explosion while only generating state sequences. Thus, we propose a technique to generate state sequences from P and check if such state sequences are accepted by S in a stratified way. A tool is developed to support the proposed technique that can be processed naturally in parallel. Some experiments demonstrate that the proposed technique mitigates the state space explosion, which cannot be achieved with the straightforward use of JPF.
By exome sequencing, we found de novo SMARCB1 mutations in two of five individuals with typical Coffin-Siris syndrome (CSS), a rare autosomal dominant anomaly syndrome. As SMARCB1 encodes a subunit ...of the SWItch/Sucrose NonFermenting (SWI/SNF) complex, we screened 15 other genes encoding subunits of this complex in 23 individuals with CSS. Twenty affected individuals (87%) each had a germline mutation in one of six SWI/SNF subunit genes, including SMARCB1, SMARCA4, SMARCA2, SMARCE1, ARID1A and ARID1B.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
In this paper, we present the findings of a study investigating the impact of shape on the taste perception of chocolate. Previous research has explored the influence of various sensory information ...on taste perception, but there has been little focus on the effect of food shape being eaten on taste perception. To explore this, we focused on the Bouba-Kiki effect, illustrating an interaction between shape and several modalities, and investigated the effect of Bouba- and Kiki-shaped (rounded and angular) foods eaten on taste perception. We utilized a 3D food printer to produce four different shapes of chocolate pieces based on the Bouba-Kiki. Participants tasted each piece and completed a chocolate flavor questionnaire. With Bayesian analysis, we determined that the Bouba-shaped chocolate pieces were perceived as sweeter than the Kiki-shaped ones, supporting earlier studies on crossmodal correspondences between shape and taste perception. However, there were no significant differences in ratings of other tastes, such as sourness and bitterness. Our research indicates that shape can affect taste perception during consumption and suggests that 3D food printers offer an opportunity to design specific shapes that influence taste experiences.
Because processes run concurrently in multitask systems, the size of the state space grows exponentially. Therefore, it is not straightforward to formally verify that such systems enjoy desired ...properties. Real-time constrains make the formal verification more challenging. In this paper, we propose the following to address the challenge: (1) a way to model multitask real-time systems as observational transition systems (OTSs), a kind of state transition systems, (2) a way to describe their specifications in CafeOBJ, an algebraic specification language, and (3) a way to verify that such systems enjoy desired properties based on such formal specifications by writing proof scores, proof plans, in CafeOBJ. As a case study, we model Fischer's protocol, a well-known real-time mutual exclusion protocol, as an OTS, describe its specification in CafeOBJ, and verify that the protocol enjoys the mutual exclusion property when an arbitrary number of processes participates in the protocol*.
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