The conformational dynamics of METTL3-METTL14 (M3/M14) heterodimer, the catalytic core for N6-methyladenosine (m6A) deposition, remain largely unexplored, limiting insight into dynamic regulation of catalysis and opportunities for therapeutic targeting in cancer. Here, we report the first single-molecule visualization of M3/M14 dynamics by high-speed atomic force microscopy (HS-AFM). Our measurements show that substrate RNA binding induces a conformational transition from a rigid apo heterodimer with an open interlobe groove to a compact, catalytically competent state. Guided by these dynamic insights, we identify two potent DNA aptamer inhibitors of M3/M14, M3B, and M3L, using a competitive in vitro selection strategy. HS-AFM integrated with molecular docking reveals that both aptamers insert into the M3/M14 interface, forming a sandwich-like complex that stabilizes a distorted, open conformation and prevents RNA-induced compaction. This conformational trapping inhibits methyltransferase activity, reduces global m6A levels, and suppresses A549 lung cancer cell growth. These findings define a dynamically regulated, interlobe targetable state of the m6A writer complex and demonstrate the utility of HS-AFM for uncovering dynamic regulatory mechanisms and guiding the development of conformationally targeted therapeutics in epitranscriptomic biology.

Data science methodologies can be applied to “molecular archeology.” By statistically inferring ancestral gene or protein sequences from a molecular phylogeny, researchers can recreate ancient molecules for laboratory experiments, allowing direct examination of their properties and structures. This review summarizes studies that applied this approach to investigate how whales and seals readapted to deep-sea diving through the evolutionary modification of myoglobins.

Polyethylene glycol (PEG) induces protein precipitation when mixed with a protein solution. This PEG-precipitation assay was developed to evaluate protein solubility while minimizing the consumption of the protein sample and has garnered increasing recognition as an exemplary method for quality assessment and screening of drug-protein candidates (e.g., antibodies) in biopharmaceutics. However, the interpretation of PEG-induced protein precipitation is complex. For example, sperm whale myoglobin and its ancestral proteins exhibit varying precipitation tolerances that cannot be adequately explained by excluded volume effects. To better understand the molecular interactions that contribute to precipitation tolerance, we introduced a semi-empirical analysis based on apparent interaction coefficients. We demonstrated that the variations observed in the precipitation tolerance of myoglobin proteins can be effectively elucidated through an analysis of protein-protein interactions.

The conformational dynamics of METTL3-METTL14 (M3/M14) heterodimer, the catalytic core for N6-methyladenosine (m6A) deposition, remain largely unexplored, limiting insight into dynamic regulation of catalysis and opportunities for therapeutic targeting in cancer. Here, we report the first single-molecule visualization of M3/M14 dynamics by high-speed atomic force microscopy (HS-AFM). Our measurements show that substrate RNA binding induces a conformational transition from a rigid apo heterodimer with an open interlobe groove to a compact, catalytically competent state. Guided by these dynamic insights, we identify two potent DNA aptamer inhibitors of M3/M14, M3B, and M3L, using a competitive in vitro selection strategy. HS-AFM integrated with molecular docking reveals that both aptamers insert into the M3/M14 interface, forming a sandwich-like complex that stabilizes a distorted, open conformation and prevents RNA-induced compaction. This conformational trapping inhibits methyltransferase activity, reduces global m6A levels, and suppresses A549 lung cancer cell growth. These findings define a dynamically regulated, interlobe targetable state of the m6A writer complex and demonstrate the utility of HS-AFM for uncovering dynamic regulatory mechanisms and guiding the development of conformationally targeted therapeutics in epitranscriptomic biology.

Human small ubiquitin‑like modifier 2 (hSUMO2) is a compact, soluble protein that can serve as a scaffold for display libraries, but systematic information on library composition and quality is limited. Here, we engineered two hSUMO2‑based phage display libraries and characterized their composition and sequence diversity by next‑generation sequencing (NGS) and affinity selection against human tumor necrosis factor‑α (TNF‑α) as a model target. Five solvent‑exposed residues (positions 56, 59, 61, 63, and 65) were randomized with NNK (Lib1) or NNB (Lib2) codons, yielding library sizes of 6 × 10⁷ and 4 × 10⁷ clones, respectively. NGS analysis indicated effective diversities of 2.6 × 10⁷ (Lib1) and 1.8 × 10⁷ (Lib2) correctly assembled variants and revealed marked position‑dependent amino acid biases, including enrichment of polar residues and depletion of hydrophobic residues, consistent with structural constraints of the SUMO2 scaffold. After one round of negative selection and six rounds of panning on TNF‑α‑immobilized magnetic beads, both libraries showed increased phage ELISA signals and enrichment of specific sequence variants, demonstrating that the constructed libraries can support affinity selection under standard process conditions. A representative mutant (TL2a) from Lib2 was expressed as a soluble protein and exhibited a detectable binding response to immobilized TNF‑α in Biacore analysis. Overall, this study provides NGS‑guided composition and quality analysis of hSUMO2 phage display libraries and practical information that may guide future engineering of SUMO‑based and other non‑antibody scaffold display systems.

Polyethylene glycol (PEG) is a widely used precipitant to concentrate proteins. The effect of PEG is generally understood to be an entropic attraction between proteins due to the depletion effect of PEG around proteins. However, measurements by Bloustine et al. [Phys. Rev. Lett. 96, 087803 (2006)] of the liquid–liquid phase separation (LLPS) temperature have shown that a lysozyme solution is stabilized and destabilized by the addition of low and high molecular-weight PEG, respectively. They also presented a theoretical model of the LLPS temperature as a virial expansion of the free energy and concluded that, in addition to the depletion effect, the attractive interaction between protein and PEG is necessary to explain the experiments. In the present study, theoretical calculations based on liquid-state density functional theory utilizing coarse-grained models are conducted to demonstrate that the protein–PEG effective attraction is responsible for the suppression and promotion of LLPS upon the addition of low- and high-weight PEG, respectively. In contrast, if the interactions between the protein and the PEG are solely due to the excluded volume effect, PEG of any molecular weight destabilizes the solution. These results suggest the necessity to reconsider the conventional understanding of the effects of polymer addition, which have been historically attributed to solely the depletion force.

Targeting abnormal dysregulation of adenosine-to-inosine deamination by ADAR enzymes offers a promising therapeutic strategy in cancer research. However, the development of effective inhibitors is impeded by the incomplete structural information on ADAR1 complexes. In this study, we employ a combination of computational 3D modeling and high-speed atomic force microscopy to elucidate the atomic and molecular dynamics of ADAR1. Two distinct interface regions (IFx and IFy) on the surface of the deaminase domain and oligomerization structural models are identified. Single-molecule-level insights into the structural dynamics of ADAR1 reveal the oligomerization of ADAR1 monomers through the self-assembly of deaminase domains. In the presence of the substrate dsRNA, the N-terminal region, including RNA-binding domains, of ADAR1 dimer exhibits a controlled flexible conformation and promotes a stable dimeric interaction with dsRNA for RNA editing. These findings provide the basis for the development of targeted inhibitors to regulate ADAR1 activity in therapeutic applications.

Onoceroids are a rare family of triterpenes. One representative onoceroid is ambrein, which is the main component of ambergris used as a traditional medicine. We have previously identified the onoceroid synthase, BmeTC, in Bacillus megaterium and succeeded in creating ambrein synthase by introducing mutations into BmeTC. Owing to the structural similarity of ambrein to vitamin D, a molecule with diverse biological activities, we hypothesized that some of the activities of ambergris may be induced by the binding of ambrein to the vitamin D receptor (VDR). We demonstrated the VDR binding ability of ambrein. By comparing the structure-activity relationships of triterpenes with both the VDR affinity and osteoclastic differentiation-promoting activity, we observed that the activity of ambrein was not induced via the VDR. Therefore, some of the activities of ambergris, but not all, can be attributed to its VDR interaction. Additionally, six unnatural onoceroids were synthesized using the BmeTC reactions, and these compounds exhibited higher VDR affinity than that of ambrein. Enzymatic syntheses of onoceroid libraries will be valuable in creating a variety of bioactive compounds beyond ambergris.

The inside of living cells is crowded by extremely high concentrations of biomolecules, and thus globular proteins should have been developed to increase their solubility under such crowding conditions during organic evolution. The O2-storage protein myoglobin (Mb) is known to be expressed in myocytes of diving mammals in much larger quantities than those of land mammals. We have previously resurrected ancient whale and pinniped Mbs and experimentally demonstrated that the diving animal Mbs have evolved to maintain high solubility under the crowding conditions or to increase their tolerance against macromolecular precipitants, rather than solubility in a dilute buffer solution. However, the detail of chemical mechanisms of the precipitant tolerance remains unclear. Here, we investigated pH dependence of the precipitant tolerance (β, slope of the solubility against precipitant concentration) of extant Mbs and plotted the β values, as well as those of ancestral Mbs, against their surface net charges (ZMb). The results demonstrated that the precipitant tolerance was approximated by the square of ZMb, that is, β = aZMb2 + b, in which a and b are constants. This effect of ZMb against the precipitation is not predicted by a classical excluded volume theory that gives constant β for Mbs but can be explained by electrostatic repulsion between Mb molecules. The present study elucidates how Mb molecules have evolved to increase their in vivo solubility and shows the physiological significance of either neutral or basic isoelectric points (pI) of the natural Mbs, rather than acidic pI.

Overexpression of the vitamin D3-inactivating enzyme CYP24A1 (cytochrome P450 family 24 subfamily and hereafter referred to as CYP24) can cause chronic kidney diseases, osteoporosis, and several types of cancers. Therefore, CYP24 inhibition has been considered a potential therapeutic approach. Vitamin D3 mimetics and small molecule inhibitors have been shown to be effective, but nonspecific binding, drug resistance, and potential toxicity limit their effectiveness. We have identified a novel 70-nt DNA aptamer-based inhibitor of CYP24 by utilizing the competition-based aptamer selection strategy, taking CYP24 as the positive target protein and CYP27B1 (the enzyme catalyzing active vitamin D3 production) as the countertarget protein. One of the identified aptamers, Apt-7, showed a 5.8-fold higher binding affinity with CYP24 than the similar competitor CYP27B1. Interestingly, Apt-7 selectively inhibited CYP24 (the relative CYP24 activity decreased by 39.1 ± 3% and showed almost no inhibition of CYP27B1). Furthermore, Apt-7 showed cellular internalization in CYP24-overexpressing A549 lung adenocarcinoma cells via endocytosis and induced endogenous CYP24 inhibition-based antiproliferative activity in cancer cells. We also employed high-speed atomic force microscopy experiments and molecular docking simulations to provide a single-molecule explanation of the aptamer-based CYP24 inhibition mechanism. The novel aptamer identified in this study presents an opportunity to generate a new probe for the recognition and inhibition of CYP24 for biomedical research and could assist in the diagnosis and treatment of cancer.

Active vitamin D form 1α,25-dihydroxtvitamin D3 (1,25(OH)2D3) plays pivotal roles in calcium homeostasis and osteogenesis via its transcription regulation effect via binding to vitamin D receptor (VDR). Mutated VDR often causes hereditary vitamin D-dependent rickets (VDDR) type II, and patients with VDDR-II are hardly responsive to physiological doses of 1,25(OH)D3. Current therapeutic approaches, including high doses of oral calcium and supraphysiologic doses of 1,25(OH)2D3, have limited success and fail to improve the quality of life of affected patients. Thus, various vitamin D analogues have been developed as therapeutic options. In our previous study, we generated genetically modified rats with mutated Vdr(R270L), an ortholog of human VDR(R274L) isolated from the patients with VDDR-II. The significant reduced affinity toward 1,25(OH)2D3 of rat Vdr(R270L) enabled us to evaluate biological activities of exogenous VDR ligand without 1α-hydroxy group such as 25(OH)D3. In this study, 2α-[2-(tetrazol-2-yl)ethyl]-1α,25(OH)2D3 (AH-1) exerted much higher affinity for Vdr(R270L) in in vitro ligand binding assay than both 25(OH)D3 and 1,25(OH)2D3. A robust osteogenic activity of AH-1 was observed in Vdr(R270L) rats. Only a 40-fold lower dose of AH-1 than that of 25(OH)D3 was effective in ameliorating rickets symptoms in Vdr(R270L) rats. Therefore, AH-1 may be promising for the therapy of VDDR-II with VDR(R274L).

Epitheaflagallin (ETFG) and epitheaflagallin 3-O-gallate (ETFGg) are minor polyphenols found in black tea extract that can be enzymatically synthesized from epigallocatechin (EGC) and epigallocatechin 3-O-gallate (EGCg), respectively, in green tea extract via laccase oxidation in the presence of gallic acid. Both compounds exhibit versatile physiological functions in vivo and in vitro, including antioxidative activity, pancreatic lipase inhibition, glycosyltransferase inhibition, and inhibition of matrix metalloprotease (MMP)-1 and MMP-3 activity and synthesis. We recently succeeded in efficiently producing ETFGg from EGCg and gallic acid by using recombinant Hericium collaroides laccase (Lcc2) in a biphasic reaction system. Using this sample, we conducted an in silico survey and in vivo tests to investigate whether ETFGg can prevent cancer based on known data showing the cancer-prevention effects of EGCg, which is a major and active component in green tea extract. We conducted docking simulation studies of the inhibitory effects of ETFGg and related compounds toward MMP-2 and MMP-9, two antiapoptotic B-cell lymphoma (Bcl)-2-family proteins, and 67-kDa laminin receptor protein (67LR). The results suggested that ETFGg interacts more strongly with Bcl-2 family proteins and 67LR compared with EGCg. Data showing the effect of ETFGg on an in vivo mouse metastasis model indicated that ETFGg is a promising functional food ingredient with potential anticancer activities. Moreover, we investigated the antidiabetic effect of ETFGg in a type-2 diabetes model mouse (KK-Ay/TaJcl) but observed no significant blood glucose inhibitory effect.

Myoglobin (Mb) is highly concentrated in the myocytes of diving mammals such as whales and seals, in comparison with land animals, and its molecular evolution has played a crucial role in their deep-sea adaptation. We previously resurrected ancestral whale Mbs and demonstrated the evolutional strategies for higher solubility under macromolecular crowding conditions. Pinnipeds, such as seals and sea lions, are also expert diving mammals with Mb-rich muscles. In the present study, we resurrected ancestral pinniped Mbs and investigated their biochemical and structural properties. Comparisons between pinniped and whale Mbs revealed the common and distinctive strategies for the deep-sea adaptation. The overall evolution processes, gaining precipitant tolerance and improving thermodynamic stability, were commonly observed. However, the strategies for improving the folding stability differed, and the pinniped Mbs exploited the shielding of hydrophobic surfaces more effectively than the whale Mbs.

Hiroshi IMAMURA1,*, Tomonari SUMI2, and Yasuhiro ISOGAI3 1 Department of Applied Chemistry, College of Life Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan 2 Research Institute for Interdisciplinary Science, Okayama University, 3-1-1 Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan. 3 Department of Pharmaceutical Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan

Hiroshi IMAMURA and Yasuhiro ISOGAI Department of Applied Chemistry, College of Life Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan

Proteins are attractive materials for supramolecular chemistry due to their multifunctionality and self-organization ability. In this work, we synthesized a diheme compound, in which two iron-protoporphyrin IX molecules are associated via a linker chain, and introduced it into a de novo designed four-helix bundle protein with two heme-binding sites. The protein gradually bound the diheme compound by bis-histidyl ligation and formed supramolecular polymers. Polymer formation was observed by atomic force microscopy (AFM), which revealed the highly branched, dendritic forms of the fibrous architecture. The present results may open a pathway toward nanowire construction with de novo heme-proteins.

Extant cetaceans, such as sperm whale, acquired the great ability to dive into the ocean depths during the evolution from their terrestrial ancestor that lived about 50 million years ago. Myoglobin (Mb) is highly concentrated in the myocytes of diving animals, in comparison with those of land animals, and is thought to play a crucial role in their adaptation as the molecular aqualung. Here, we resurrected ancestral whale Mbs, which are from the common ancestor between toothed and baleen whales (Basilosaurus), and from a further common quadrupedal ancestor between whale and hippopotamus (Pakicetus). The experimental and theoretical analyses demonstrated that whale Mb adopted two distinguished strategies to increase the protein concentration in vivo along the evolutionary history of deep sea adaptation; gaining precipitant tolerance in the early phase of the evolution, and increase of folding stability in the late phase.

Epitheaflagallin (ETFG) and epitheaflagallin 3-O-gallate (ETFGg) are minor polyphenols in black tea extract that are enzymatically synthesized from epigallocatechin (EGC) and epigallocatechin gallate (EGCg), respectively, in green tea extract via laccase oxidation in the presence of gallic acid. The constituents of laccase-treated green tea extract in the presence of gallic acid are thus quite different from those of nonlaccase-treated green tea extract: EGC and EGCg are present in lower concentrations, and ETFG and ETFGg are present in higher concentrations. Additionally, laccase-treated green tea extract contains further polymerized catechin derivatives, comparable with naturally fermented teas such as oolong tea and black tea. We found that ETFGg and laccase-treated green tea extracts exhibit versatile physiological functions in vivo and in vitro, including antioxidative activity, pancreatic lipase inhibition, Streptococcus sorbinus glycosyltransferase inhibition, and an inhibiting effect on the activity of matrix metalloprotease-1 and -3 and their synthesis by human gingival fibroblasts. We confirmed that these inhibitory effects of ETFGg in vitro match well with the results obtained by docking simulations of the compounds with their target enzymes or noncatalytic protein. Thus, ETFGg and laccase-treated green tea extracts containing ETFGg are promising functional food materials with potential antiobesity and antiperiodontal disease activities.