Isolation and Structure Elucidation of JBIR-157, a Skeletally Novel Aromatic Polyketide Produced by the Heterologous Expression of a Cryptic Gene Cluster.

Heterologous expression of natural compound biosynthetic gene clusters (BGCs) is a robust approach for not only revealing the biosynthetic mechanisms leading to the compounds, but also for discovering new products from uncharacterized BGCs. We established a heterologous expression technique applicable to huge biosynthetic gene clusters for generating large molecular secondary metabolites such as type-I polyketides. As an example, we targeted concanamycin BGC from Streptomyces neyagawaensis IFO13477 (the cluster size of 99 kbp), and obtained a bacterial artificial chromosome (BAC) clone with an insert size of 211 kbp that contains the entire concanamycin BGC. Interestingly, heterologous expression for this BAC clone resulted in two additional aromatic polyketides, ent-gephyromycin, and a new compound designated as JBIR-157, together with the expected concanamycin. Bioinformatic and biochemical analyses revealed that a cryptic biosynthetic gene cluster in this BAC clone was responsible for the production of these type-II polyketide synthases (PKS) compounds. Here, we describe the production, isolation, and structure elucidation of JBIR-157, determined primarily by a series of NMR spectral analyses.

A high dose KRP203 induces cytoplasmic vacuoles associated with altered phosphoinositide segregation and endosome expansion.

In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE and VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies.

A high-resolution structural characterization and physicochemical study of how a peptoid binds to an oncoprotein MDM2.

Peptoids are a promising drug modality targeting disease-related proteins, but how a peptoid engages in protein binding is poorly understood. This is primarily due to a lack of high-resolution peptoid-protein complex structures and systematic physicochemical studies. Here, we present the first crystal structure of a peptoid bound to a protein, providing high-resolution structural information about how a peptoid binds to a protein. We previously reported a rigid peptoid, oligo(N-substituted alanine) (oligo-NSA), and developed an oligo-NSA-type peptoid that binds to MDM2. X-ray crystallographic analysis of the peptoid bound to MDM2 showed that the peptoid recognizes the MDM2 surface predominantly through the interaction of the N-substituents, while the main chain acts as a scaffold. Additionally, conformational, thermodynamic, and kinetic analysis of the peptoid and its derivatives with a less rigid main chain revealed that rigidification of the peptoid main chain contributes to improving the protein binding affinity. This improvement is thermodynamically attributed to an increased magnitude of the binding enthalpy change, and kinetically to an increased association rate and decreased dissociation rate. This study provides invaluable insights into the design of protein-targeting peptoids.

Molecular basis promoting centriole triplet microtubule assembly.

The triplet microtubule, a core structure of centrioles crucial for the organization of centrosomes, cilia, and flagella, consists of unclosed incomplete microtubules. The mechanisms of its assembly represent a fundamental open question in biology. Here, we discover that the ciliopathy protein HYLS1 and the ß-tubulin isotype TUBB promote centriole triplet microtubule assembly. HYLS1 or a C-terminal tail truncated version of TUBB generates tubulin-based superstructures composed of centriole-like incomplete microtubule chains when overexpressed in human cells. AlphaFold-based structural models and mutagenesis analyses further suggest that the ciliopathy-related residue D211 of HYLS1 physically traps the wobbling C-terminal tail of TUBB, thereby suppressing its inhibitory role in the initiation of the incomplete microtubule assembly. Overall, our findings provide molecular insights into the biogenesis of atypical microtubule architectures conserved for over a billion years.

Estimating Counterfactual Treatment Outcomes Over Time in Complex Multiagent Scenarios.

Evaluation of intervention in a multiagent system, for example, when humans should intervene in autonomous driving systems and when a player should pass to teammates for a good shot, is challenging in various engineering and scientific fields. Estimating the individual treatment effect (ITE) using counterfactual long-term prediction is practical to evaluate such interventions. However, most of the conventional frameworks did not consider the time-varying complex structure of multiagent relationships and covariate counterfactual prediction. This may lead to erroneous assessments of ITE and difficulty in interpretation. Here, we propose an interpretable, counterfactual recurrent network in multiagent systems to estimate the effect of the intervention. Our model leverages graph variational recurrent neural networks (GVRNNs) and theory-based computation with domain knowledge for the ITE estimation framework based on long-term prediction of multiagent covariates and outcomes, which can confirm the circumstances under which the intervention is effective. On simulated models of an automated vehicle and biological agents with time-varying confounders, we show that our methods achieved lower estimation errors in counterfactual covariates and the most effective treatment timing than the baselines. Furthermore, using real basketball data, our methods performed realistic counterfactual predictions and evaluated the counterfactual passes in shot scenarios.

Multimodal action of KRP203 on phosphoinositide kinases in vitro and in cells.

Increased phosphoinositide signaling is commonly associated with cancers. While "one-drug one-target" has been a major drug discovery strategy for cancer therapy, a "one-drug multi-targets" approach for phosphoinositide enzymes has the potential to offer a new therapeutic approach. In this study, we sought a new way to target phosphoinositides metabolism. Using a high-throughput phosphatidylinositol 5-phosphate 4-kinase-alpha (PI5P4Kα) assay, we have identified that the immunosuppressor KRP203/Mocravimod induces a significant perturbation in phosphoinositide metabolism in U87MG glioblastoma cells. Despite high sequence similarity of PI5P4K and PI4K isozymes, in vitro kinase assays showed that KRP203 activates some (e.g., PI5P4Kα, PI4KIIß) while inhibiting other phosphoinositide kinases (e.g., PI5P4Kß, γ, PI4KIIα, class I PI3K-p110α, δ, γ). Furthermore, KRP203 enhances PI3P5K/PIKFYVE's substrate selectivity for phosphatidylinositol (PI) while preserving its selectivity for PI(3)P. At cellular levels, 3 h of KRP203 treatment induces a prominent increase of PI(3)P and moderate increase of PI(5)P, PI(3,5)P2, and PI(3,4,5)P3 levels in U87MG cells. Collectively, the finding of multimodal activity of KRP203 towards multi-phosphoinositide kinases may open a novel basis to modulate cellular processes, potentially leading to more effective treatments for diseases associated with phosphoinositide signaling pathways.

The oxidative folding of nascent polypeptides provides electrons for reductive reactions in the ER.

The endoplasmic reticulum (ER) maintains an oxidative redox environment that is advantageous for the oxidative folding of nascent polypeptides entering the ER. Reductive reactions within the ER are also crucial for maintaining ER homeostasis. However, the mechanism by which electrons are supplied for the reductase activity within the ER remains unknown. Here, we identify ER oxidoreductin-1α (Ero1α) as an electron donor for ERdj5, an ER-resident disulfide reductase. During oxidative folding, Ero1α catalyzes disulfide formation in nascent polypeptides through protein disulfide isomerase (PDI) and then transfers the electrons to molecular oxygen via flavin adenine dinucleotide (FAD), ultimately yielding hydrogen peroxide (H2O2). Besides this canonical electron pathway, we reveal that ERdj5 accepts electrons from specific cysteine pairs in Ero1α, demonstrating that the oxidative folding of nascent polypeptides provides electrons for reductive reactions in the ER. Moreover, this electron transfer pathway also contributes to maintaining ER homeostasis by reducing H2O2 production in the ER.

Nonthermal acceleration of protein hydration by sub-terahertz irradiation.

The collective intermolecular dynamics of protein and water molecules, which overlap in the sub-terahertz (THz) frequency region, are relevant for expressing protein functions but remain largely unknown. This study used dielectric relaxation (DR) measurements to investigate how externally applied sub-THz electromagnetic fields perturb the rapid collective dynamics and influence the considerably slower chemical processes in protein-water systems. We analyzed an aqueous lysozyme solution, whose hydration is not thermally equilibrated. By detecting time-lapse differences in microwave DR, we demonstrated that sub-THz irradiation gradually decreases the dielectric permittivity of the lysozyme solution by reducing the orientational polarization of water molecules. Comprehensive analysis combining THz and nuclear magnetic resonance spectroscopies suggested that the gradual decrease in the dielectric permittivity is not induced by heating but is due to a slow shift toward the hydrophobic hydration structure in lysozyme. Our findings can be used to investigate hydration-mediated protein functions based on sub-THz irradiation.

Functional molecular evolution of a GTP sensing kinase: PI5P4Kß.

Over 4 billion years of evolution, multiple mutations, including nucleotide substitutions, gene and genome duplications and recombination, have established de novo genes that translate into proteins with novel properties essential for high-order cellular functions. However, molecular processes through which a protein evolutionarily acquires a novel function are mostly speculative. Recently, we have provided evidence for a potential evolutionary mechanism underlying how, in mammalian cells, phosphatidylinositol 5-phosphate 4-kinase ß (PI5P4Kß) evolved into a GTP sensor from ATP-utilizing kinase. Mechanistically, PI5P4Kß has acquired the guanine efficient association (GEA) motif by mutating its nucleotide base recognition sequence, enabling the evolutionary transition from an ATP-dependent kinase to a distinct GTP/ATP dual kinase with its KM for GTP falling into physiological GTP concentrations-the genesis of GTP sensing activity. Importantly, the GTP sensing activity of PI5P4Kß is critical for the manifestation of cellular metabolism and tumourigenic activity in the multicellular organism. The combination of structural, biochemical and biophysical analyses used in our study provides a novel framework for analysing how a protein can evolutionarily acquire a novel activity, which potentially introduces a critical function to the cell.

Amide-to-ester substitution as a stable alternative to N-methylation for increasing membrane permeability in cyclic peptides.

Naturally occurring peptides with high membrane permeability often have ester bonds on their backbones. However, the impact of amide-to-ester substitutions on the membrane permeability of peptides has not been directly evaluated. Here we report the effect of amide-to-ester substitutions on the membrane permeability and conformational ensemble of cyclic peptides related to membrane permeation. Amide-to-ester substitutions are shown to improve the membrane permeability of dipeptides and a model cyclic hexapeptide. NMR-based conformational analysis and enhanced sampling molecular dynamics simulations suggest that the conformational transition of the cyclic hexapeptide upon membrane permeation is differently influenced by an amide-to-ester substitution and an amide N-methylation. The effect of amide-to-ester substitution on membrane permeability of other cyclic hexapeptides, cyclic octapeptides, and a cyclic nonapeptide is also investigated to examine the scope of the substitution. Appropriate utilization of amide-to-ester substitution based on our results will facilitate the development of membrane-permeable peptides.

Helix-forming aliphatic homo-δ-peptide foldamers based on the conformational restriction effects of cyclopropane.

Considerable effort has been directed toward developing artificial peptide-based foldamers. However, detailed structural analysis of δ-peptide foldamers consisting of only aliphatic δ-amino acids has not been reported. Herein, we rationally designed and stereoselectively synthesized aliphatic homo-δ-peptides forming a stable helical structure by using a chiral cyclopropane δ-amino acid as a monomer unit. Structural analysis of the homo-δ-peptides using circular dichroism, infrared, and NMR spectroscopy indicated that they form a stable 14-helical structure in solution. Furthermore, we successfully conducted X-ray crystallographic analysis of the homo-δ-peptides, demonstrating a right-handed 14-helical structure. This helical structure of the crystal was consistent with those predicted by theoretical calculations and those obtained based on NMR spectroscopy in solution. This stable helical structure is due to the effective restriction of the backbone conformation by the structural characteristics of cyclopropane. This work reports the first example of aliphatic homo-δ-peptide foldamers having a stable helical structure both in the solution and crystal states.

Absence of ULK1 decreases AMPK activity in the kidney, leading to chronic kidney disease progression.

AMP-activated protein kinase (AMPK) inactivation in chronic kidney disease (CKD) leads to energy status deterioration in the kidney, constituting the vicious cycle of CKD exacerbation. Unc-51-like kinase 1 (ULK1) is considered a downstream molecule of AMPK; however, it was recently reported that the activity of AMPK could be regulated by ULK1 conversely. We demonstrated that AMPK and ULK1 activities were decreased in the kidneys of CKD mice. However, whether and how ULK1 is involved in the underlying mechanism of CKD exacerbation remains unknown. In this study, we investigated the ULK1 involvement in CKD, using ULK1 knockout mice. The CKD model of Ulk1-/- mice exhibited significantly exacerbated renal function and worsening renal fibrosis. In the kidneys of the CKD model of Ulk1-/- mice, reduced AMPK and its downstream ß-oxidation could be observed, leading to an energy deficit of increased AMP/ATP ratio. In addition, AMPK signaling in the kidney was reduced in control Ulk1-/- mice with normal renal function compared to control wild-type mice, suggesting that ULK1 deficiency suppressed AMPK activity in the kidney. This study is the first to present ULK1 as a novel therapeutic target for CKD treatment, which regulates AMPK activity in the kidney.

15N-Detected TROSY NMR experiments to study large disordered proteins in high-field magnets.

Intrinsically disordered regions (IDRs) of proteins are critical in the regulation of biological processes but difficult to study structurally. Nuclear magnetic resonance (NMR) is uniquely equipped to provide structural information on IDRs at atomic resolution; however, existing NMR methods often pose a challenge for large molecular weight IDRs. Resonance assignment of IDRs using 15ND-detection was previously demonstrated and shown to overcome some of these limitations. Here, we improve the methodology by overcoming the need for deuterated buffers and provide better sensitivity and resolution at higher magnetic fields and physiological salt concentrations using transverse relaxation optimized spectroscopy (TROSY). Finally, large disordered regions with low sequence complexity can be assigned efficiently using these new methods as demonstrated by achieving near complete assignment of the 398-residue N-terminal IDR of the transcription factor NFAT1 harboring 18% prolines.

Residue-based program of a ß-peptoid twisted strand shape via a cyclopentane constraint.

N-Substituted peptides, such as peptoids and ß-peptoids, have been reported to have unique structures with diverse functions, like catalysis and manipulation of biomolecular functions. Recently, the preorganization of monomer shape by restricting bond rotations about all backbone dihedral angles has been demonstrated to be useful for de novo design of peptoid structures. Such design strategies are hitherto unexplored for ß-peptoids; to date, no preorganized ß-peptoid monomers have been reported. Here, we report the first design strategy for ß-peptoids, in which all four backbone dihedral angles (ω, ϕ, θ, ψ) are rotationally restricted on a per-residue basis. The introduction of a cyclopentane constraint realized the preorganized monomer structure and led to a ß-peptoid with a stable twisted strand shape.

The GTP responsiveness of PI5P4Kß evolved from a compromised trade-off between activity and specificity.

Unlike most kinases, phosphatidylinositol 5-phosphate 4-kinase ß (PI5P4Kß) utilizes GTP as a physiological phosphate donor and regulates cell growth under stress (i.e., GTP-dependent stress resilience). However, the genesis and evolution of its GTP responsiveness remain unknown. Here, we reveal that PI5P4Kß has acquired GTP preference by generating a short dual-nucleotide-recognizing motif called the guanine efficient association (GEA) motif. Comparison of nucleobase recognition with 660 kinases and 128 G proteins has uncovered that most kinases and PI5P4Kß use their main-chain atoms for adenine recognition, while the side-chain atoms are required for guanine recognition. Mutational analysis of the GEA motif revealed that the acquisition of GTP reactivity is accompanied by an extended activity toward inosine triphosphate (ITP) and xanthosine triphosphate (XTP). Along with the evolutionary analysis data that point to strong negative selection of the GEA motif, these results suggest that the GTP responsiveness of PI5P4Kß has evolved from a compromised trade-off between activity and specificity, underpinning the development of the GTP-dependent stress resilience.

Role of the Orphan Transporter SLC35E1 in the Nuclear Egress of Herpes Simplex Virus 1.

This study developed a system consisting of two rounds of screening cellular proteins involved in the nuclear egress of herpes simplex virus 1 (HSV-1). Using this system, we first screened cellular proteins that interacted with the HSV-1 nuclear egress complex (NEC) consisting of UL34 and UL31 in HSV-1-infected cells, which are critical for the nuclear egress of HSV-1, by tandem affinity purification coupled with mass spectrometry-based proteomics technology. Next, we performed CRISPR/Cas9-based screening of live HSV-1-infected reporter cells under fluorescence microscopy using single guide RNAs targeting the cellular proteins identified in the first proteomic screening to detect the mislocalization of the lamin-associated protein emerin, which is a phenotype for defects in HSV-1 nuclear egress. This study focused on a cellular orphan transporter SLC35E1, one of the cellular proteins identified by the screening system. Knockout of SLC35E1 reduced HSV-1 replication and induced membranous invaginations containing perinuclear enveloped virions (PEVs) adjacent to the nuclear membrane (NM), aberrant accumulation of PEVs in the perinuclear space between the inner and outer NMs and the invagination structures, and mislocalization of the NEC. These effects were similar to those of previously reported mutation(s) in HSV-1 proteins and depletion of cellular proteins that are important for HSV-1 de-envelopment, one of the steps required for HSV-1 nuclear egress. Our newly established screening system enabled us to identify a novel cellular protein required for efficient HSV-1 de-envelopment. IMPORTANCE The identification of cellular protein(s) that interact with viral effector proteins and function in important viral procedures is necessary for enhancing our understanding of the mechanics of various viral processes. In this study, we established a new system consisting of interactome screening for the herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), followed by loss-of-function screening to target the identified putative NEC-interacting cellular proteins to detect a defect in HSV-1 nuclear egress. This newly established system identified SLC35E1, an orphan transporter, as a novel cellular protein required for efficient HSV-1 de-envelopment, providing an insight into the mechanisms involved in this viral procedure.

Oligo(N-methylalanine) as a Peptide-Based Molecular Scaffold with a Minimal Structure and High Density of Functionalizable Sites.

Functionalizable synthetic molecules with nanometer sizes and defined shapes in water are useful as molecular scaffolds to mimic the functions of biomacromolecules and develop chemical tools for manipulating biomacromolecules. Herein, we propose oligo(N-methylalanine) (oligo-NMA) as a peptide-based molecular scaffold with a minimal structure and a high density of functionalizable sites. Oligo-NMA forms a defined shape in water without hydrogen-bonding networks or ring constraints, which enables the molecule to act as a scaffold with minimal atomic composition. Furthermore, functional groups can be readily introduced on the nitrogens and α-carbons of oligo-NMA. Computational and NMR spectroscopic analysis suggested that the backbone structure of oligo-NMA is not largely affected by functionalization. Moreover, the usefulness of oligo-NMA was demonstrated by the design of protein ligands. The ease of synthesis, minimal structure, and high functionalization flexibility makes oligo-NMA a useful scaffold for chemical and biological applications.

3CL Protease Inhibitors with an Electrophilic Arylketone Moiety as Anti-SARS-CoV-2 Agents.

The novel coronavirus, SARS-CoV-2, has been identified as the causative agent for the current coronavirus disease (COVID-19) pandemic. 3CL protease (3CLpro) plays a pivotal role in the processing of viral polyproteins. We report peptidomimetic compounds with a unique benzothiazolyl ketone as a warhead group, which display potent activity against SARS-CoV-2 3CLpro. The most potent inhibitor YH-53 can strongly block the SARS-CoV-2 replication. X-ray structural analysis revealed that YH-53 establishes multiple hydrogen bond interactions with backbone amino acids and a covalent bond with the active site of 3CLpro. Further results from computational and experimental studies, including an in vitro absorption, distribution, metabolism, and excretion profile, in vivo pharmacokinetics, and metabolic analysis of YH-53 suggest that it has a high potential as a lead candidate to compete with COVID-19.

Predicting anesthetic infusion events using machine learning.

Recently, research has been conducted to automatically control anesthesia using machine learning, with the aim of alleviating the shortage of anesthesiologists. In this study, we address the problem of predicting decisions made by anesthesiologists during surgery using machine learning; specifically, we formulate a decision making problem by increasing the flow rate at each time point in the continuous administration of analgesic remifentanil as a supervised binary classification problem. The experiments were conducted to evaluate the prediction performance using six machine learning models: logistic regression, support vector machine, random forest, LightGBM, artificial neural network, and long short-term memory (LSTM), using 210 case data collected during actual surgeries. The results demonstrated that when predicting the future increase in flow rate of remifentanil after 1 min, the model using LSTM was able to predict with scores of 0.659 for sensitivity, 0.732 for specificity, and 0.753 for ROC-AUC; this demonstrates the potential to predict the decisions made by anesthesiologists using machine learning. Furthermore, we examined the importance and contribution of the features of each model using Shapley additive explanations-a method for interpreting predictions made by machine learning models. The trends indicated by the results were partially consistent with known clinical findings.

Function-Related Dynamics in Multi-Spanning Helical Membrane Proteins Revealed by Solution NMR.

A primary biological function of multi-spanning membrane proteins is to transfer information and/or materials through a membrane by changing their conformations. Therefore, particular dynamics of the membrane proteins are tightly associated with their function. The semi-atomic resolution dynamics information revealed by NMR is able to discriminate function-related dynamics from random fluctuations. This review will discuss several studies in which quantitative dynamics information by solution NMR has contributed to revealing the structural basis of the function of multi-spanning membrane proteins, such as ion channels, GPCRs, and transporters.