The activation of the insulin receptor (IR) is central to the regulation of physiological metabolism, growth, and proliferation, and is associated with various cancers, including colorectal cancer (CRC). Among the tyrosine residues in the intracellular kinase domain of IR, Tyr1150 plays a pivotal role in receptor activation by regulating substrate binding to the kinase active site. In this study, we introduce a novel approach for selectively modulating insulin signaling in CRC through the development of an intrabody targeting phosphorylated Tyr1150 (IR pY1150). Using phage display technology, we isolated a phosphorylation site-specific single-chain variable fragment (scFv), K109.1, from a human scFv antibody library, which specifically binds to IR pY1150. K109.1 was subsequently engineered as an intrabody designed to function within cells. Ectopic expression of K109.1 in CRC cells selectively inhibited insulin-mediated phosphorylation of key downstream effectors, including insulin receptor substrates and Akt, thereby leading to a significant reduction in insulin-dependent glucose uptake. Notably, K109.1 did not affect extracellular signal-regulated kinase phosphorylation or alter cell proliferation, migration, or invasion. We further evaluated K109.1 in BT-474 and HEK293 cells to assess its effects in additional cellular models. In BT-474 breast cancer cells, K109.1 selectively inhibited Akt phosphorylation, while in HEK293 cells it suppressed both Akt and ERK phosphorylation, indicating context-specific signaling responses. Taken together, these findings indicate that intrabody-mediated targeting of IR pY1150 is crucial for regulating glucose metabolism, suggesting that the developed antibody, K109.1, could serve as a tool for modulating insulin-mediated signaling pathways.

Insulin binding to the insulin receptor (IR) triggers signaling pathways that regulate glucose uptake and cell growth. In previous work, we identified a DNA aptamer, A62, which partially activates the IR. During engineering aptamers for improved in vivo stability, we discovered that crosslinking two A62 aptamers with linkers of varying lengths led to full phosphorylation of the IR, although activation remained selective to the AKT pathway. Here, to elucidate the mechanism behind this aptamer-induced full activation of the IR, we determined the structure of the IR in complex with a dimeric form of A62 (A62D) linked by an eight-nucleotide connector. We identified three distinct conformations of the IR: arrowhead-shaped, pseudo-arrowhead-shaped and pseudo-gamma-shaped. The pseudo-gamma-shaped conformation closely resembles the structure of a fully active IR bound by a single insulin molecule. In these configurations, only one A62 monomer (A62M) within the A62D dimer binds to the IR dimer. This binding brings the IR monomers into close proximity, promoting intermolecular trans-phosphorylation. Our findings provide valuable structural insights for the development of novel therapeutic strategies targeting the IR.

Organic dyes play a crucial role in live-cell imaging because of their advantageous properties, such as photostability and high brightness. Here we introduce a super-photostable and bright organic dye, Phoenix Fluor 555 (PF555), which exhibits an order-of-magnitude longer photobleaching lifetime than conventional organic dyes without the requirement of any anti-photobleaching additives. PF555 is an asymmetric cyanine structure in which, on one side, the indole in the conventional Cyanine-3 is substituted with 3-oxo-quinoline. PF555 provides a powerful tool for long-term live-cell single-molecule imaging, as demonstrated by the imaging of the dynamic single-molecule interactions of the epidermal growth factor receptor with clathrin-coated structures on the plasma membrane of a live cell under physiological conditions. Phoenix Fluor 555 (PF555) is a bright dye with an exceptional, order-of-magnitude longer photobleaching lifetime than conventional organic dyes that enables extended live-cell single-molecule imaging without anti-photobleaching additives.

Signal transduction by the human epidermal growth factor receptor (EGFR) is modulated by its ligands as well as by the various compounds on the plasma membrane (PM), including cholesterol. Here, using single-molecule coimmunoimmobilization, we found that cholesterol in the inner leaflet of PM plays an essential role in ligand-dependent EGFR activation. This was done by direct and quantitative examination of the interaction between EGFR and cholesterol on the PM of a living cell. Structural analysis using molecular dynamics simulations and nuclear magnetic resonance spectroscopy further revealed that cholesterol in the inner leaflet is critically important for the transition of the dimeric transmembrane domain (TMD) of the receptor to the active conformation. We also identified the specific pattern of residues Ile640-Gly641-Leu642 of EGFR TMD regulating inner leaflet cholesterol binding and ligand-activated EGFR by screening via site-directed mutagenesis. Our results indicate that cholesterol in the inner leaflet of PM is a key checkpoint of EGFR activation triggered by EGF.

The binding of insulin to the insulin receptor (IR) triggers a cascade of receptor conformational changes and autophosphorylation, leading to the activation of metabolic and mitogenic pathways. Recent advances in the structural and functional analyses of IR have revealed the conformations of the extracellular domains of the IR in inactive and fully activated states. However, the early activation mechanisms of this receptor remain poorly understood. The structures of partially activated IR in complex with aptamers provide clues for understanding the initial activation mechanism. In this review, we discuss the structural and functional features of IR complexed with various ligands and propose a model to explain the sequential activation mechanism. Moreover, we discuss the structures of IR complexed with biased agonists that selectively activate metabolic pathways and provide insights into the design of selective agonists and their clinical implications.

Phospholipase D (PLD) hydrolyzes phosphatidylcholine into phosphatidic acid (PA) and free choline. Two mammalian PLD isoforms, PLD1 and PLD2, play essential roles in the regulation of infectious and inflammatory human diseases. Many innate immune cells including neutrophils and macrophages express both PLD isoforms, and activation of PLD elicits leukocyte migration, phagocytosis, and generation of reactive oxygen species to increase innate defense activity. However, PLD2 activation blocks neutrophil extracellular trap formation, suggesting nuanced roles for PLD in cellular activities. Indeed, in addition to their normal physiologic roles, PLD1 and/or PLD2 also contribute to the pathomechanism of various human inflammatory diseases. Many studies have demonstrated that PLD exacerbates pathogenic inflammatory responses present in various respiratory, cardiovascular, retinal, intestinal, metabolic, and autoimmune diseases. Thus, PLD isoforms are attractive, druggable enzymatic mediators that hold great promise as targets for therapeutic intervention in many human infectious and inflammatory diseases.

Activation of insulin receptor (IR) initiates a cascade of conformational changes and autophosphorylation events. Herein, we determined three structures of IR trapped by aptamers using cryo-electron microscopy. The A62 agonist aptamer selectively activates metabolic signaling. In the absence of insulin, the two A62 aptamer agonists of IR adopt an insulin-accessible arrowhead conformation by mimicking site-1/site-2' insulin coordination. Insulin binding at one site triggers conformational changes in one protomer, but this movement is blocked in the other protomer by A62 at the opposite site. A62 binding captures two unique conformations of IR with a similar stalk arrangement, which underlie Tyr1150 mono-phosphorylation (m-pY1150) and selective activation for metabolic signaling. The A43 aptamer, a positive allosteric modulator, binds at the opposite side of the insulin-binding module, and stabilizes the single insulin-bound IR structure that brings two FnIII-3 regions into closer proximity for full activation. Our results suggest that spatial proximity of the two FnIII-3 ends is important for m-pY1150, but multi-phosphorylation of IR requires additional conformational rearrangement of intracellular domains mediated by coordination between extracellular and transmembrane domains.

Membrane nanotubes or tunneling nanotubes (TNTs) that connect cells have been recognized as a previously unidentified pathway for intercellular transport between distant cells. However, it is unknown how this delicate structure, which extends over tens of micrometers and remains robust for hours, is formed. Here, we found that a TNT develops from a double filopodial bridge (DFB) created by the physical contact of two filopodia through helical deformation of the DFB. The transition of a DFB to a close-ended TNT is most likely triggered by disruption of the adhesion of two filopodia by mechanical energy accumulated in a twisted DFB when one of the DFB ends is firmly attached through intercellular cadherin-cadherin interactions. These studies pinpoint the mechanistic questions about TNTs and elucidate a formation mechanism.

Phospholipase D2 (PLD2), a signaling protein, plays a central role in cellular communication and various biological processes. Here, we show that PLD2 contributes to bone homeostasis by regulating bone resorption through osteoclastic cell migration and microtubule-dependent cytoskeletal organization. Pld2 -deficient mice exhibited a low bone mass attributed to increased osteoclast function without altered osteoblast activity. While Pld2 deficiency did not affect osteoclast differentiation, its absence promoted the migration of osteoclast lineage cells through a mechanism involving M-CSF-induced activation of the PI3K–Akt–GSK3β signaling pathway. The absence of Pld2 also boosted osteoclast spreading and actin ring formation, resulting in elevated bone resorption. Furthermore, Pld2 deletion increased microtubule acetylation and stability, which were later restored by treatment with a specific inhibitor of Akt, an essential molecule for microtubule stabilization and osteoclast bone resorption activity. Interestingly, PLD2 interacted with the M-CSF receptor (c-Fms) and PI3K, and the association between PLD2 and c-Fms was reduced in response to M-CSF. Altogether, our findings indicate that PLD2 regulates bone homeostasis by modulating osteoclastic cell migration and microtubule stability via the M-CSF-dependent PI3K–Akt–GSK3β axis.

Aptamers are widely used as binders that interact with targets with high affinity or as inhibitors of the function of target molecules. However, they have also been used to modulate target protein function, which they achieve by activating the target or stabilizing its conformation. Here, we report a unique aptamer modulator of the insulin receptor (IR), IR-A62. Alone, IR-A62 acts as a biased agonist that preferentially induces Y1150 monophosphorylation of IR. However, when administered alongside insulin, IR-A62 shows variable binding cooperativity depending on the ligand concentration. At low concentrations, IR-A62 acts as a positive allosteric modulator (PAM) agonist that enhances insulin binding, but at high concentrations, it acts as a negative allosteric modulator (NAM) agonist that competes with insulin for IR. Moreover, the concentration of insulin affects the binding of IR-A62 to IR. Finally, the subcutaneous administration of IR-A62 to diabetic mice reduces blood glucose levels with a longer-lasting effect than insulin administration. These findings imply that aptamers can elicit various responses from receptors beyond those of a simple agonist or inhibitor. We expect further studies of IR-A62 to help reveal the mechanism of IR activation and greatly expand the range of therapeutic applications of aptamers.

Aminoacyl-tRNA synthetases (ARSs) have evolved to acquire various additional domains. These domains allow ARSs to communicate with other cellular proteins in order to promote non-translational functions. Vertebrate cytoplasmic isoleucyl-tRNA synthetases (IARS1s) have an uncharacterized unique domain, UNE-I. Here, we present the crystal structure of the chicken IARS1 UNE-I complexed with glutamyl-tRNA synthetase 1 (EARS1). UNE-I consists of tandem ubiquitin regulatory X (UBX) domains that interact with a distinct hairpin loop on EARS1 and protect its neighboring proteins in the multi-synthetase complex from degradation. Phosphomimetic mutation of the two serine residues in the hairpin loop releases IARS1 from the complex. IARS1 interacts with BRCA1 in the nucleus, regulates its stability by inhibiting ubiquitylation via the UBX domains, and controls DNA repair function.

Background: Accumulating evidences indicate that AXL overexpression or activation is associated with cancer progression and acquired resistance to targeted anti-cancer drugs such as epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). Despite recent development of several drugs that target multiple receptor tyrosine kinases (RTKs), drugs that selectively target AXL signaling are extremely rare. Short nucleic acid aptamers are non-immunogenic molecules with high binding affinity and specificity to their target molecules that could potentially be used as a novel cancer treatment. Methods: Modified-DNA aptamers were selected on the basis of its ability to bind recombinant human AXL. AXL aptamers were selected for their inhibition of AXL and then selected aptamers were tested for their use to overcome acquired resistant to EGFR-TKI on a lung cancer cell with acquired resistance to erlotinib. Results: These new AXL aptamers inhibited cell viability to an extent of 30–40% in HCC827/ER cells with acquired resistance to erlotinib. The possible mechanism of overcoming the acquired resistance may be by inhibiting the activation of Akt and Erk. Although, aptamers effectively decreased cell viability of erlotinib‐resistant cell line, the combination of aptamers and erlotinib did not synergistically decrease the survival of the resistant cell line. Conclusions: We developed newly modified DNA aptamers that selectively bind to AXL receptors, and assessed their efficacy in a human lung cancer cell with acquired resistance to EGFR-TKI.

Single-molecule localization microscopy (SMLM) has allowed the observation of various molecular structures in cells beyond the diffraction limit using organic dyes. In principle, the SMLM resolution depends on the precision of photoswitching fluorophore localization, which is inversely correlated with the square root of the number of photons released from the individual fluorophores. Thus, increasing the photon number by using highly bright fluorophores, such as quantum dots (QDs), can theoretically fundamentally overcome the current resolution limit of SMLM. However, the use of QDs in SMLM has been challenging because QDs have no photoswitching property, which is essential for SMLM, and they exhibit nonspecificity and multivalency, which complicate their use in fluorescence imaging. Here, we present a method to utilize QDs in SMLM to surpass the resolution limit of the current SMLM utilizing organic dyes. We confer monovalency, specificity, and photoswitchability on QDs by steric exclusion via passivation and ligand exchange with ptDNA, PEG, and casein as well as by DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) via automatic thermally driven hybridization between target-bound docking and dye-bound complementary imager strands. QDs are made monovalent and photoswitchable to enable SMLM and show substantially better photophysical properties than Cy3, with higher fluorescence intensity and an improved resolution factor. QD-PAINT displays improved spatial resolution with a narrower full width at half maximum (FWHM) than DNA-PAINT with Cy3. In summary, QD-PAINT shows great promise as a next-generation SMLM method for overcoming the limited resolution of the current SMLM.

Lipids on the plasma membrane are not only components of the membrane biophysical structures but also regulators of receptor functions. Recently, the critical roles of lipid-protein interactions have been intensively highlighted. Epidermal growth factor receptor (EGFR) is one of the most extensively studied receptors exhibiting various lipid interactions, including interactions with phosphatidylcholine, phosphatidylserine, phosphatidylinositol phosphate, cholesterol, gangliosides, and palmitate. Here, we review recent findings on how direct interaction with these lipids regulates EGFR activation and signaling, providing unprecedented insight into the comprehensive roles of various lipids in the control of EGFR functions. Finally, the current limitations in investigating lipid-protein interactions and novel technologies to potentially overcome these limitations are discussed.

Various repertoires of membrane protein interactions determine cellular responses to diverse environments around cells dynamically in space and time. Current assays, however, have limitations in unraveling these interactions in the physiological states in a living cell due to the lack of capability to probe the transient nature of these interactions on the crowded membrane. Here, we present a simple and robust assay that enables the investigation of transient protein interactions in living cells by using the single-molecule diffusional mobility shift assay (smDIMSA). Utilizing smDIMSA, we uncovered the interaction profile of EGFR with various membrane proteins and demonstrated the promiscuity of these interactions depending on the cancer cell line. The transient interaction profile obtained by smDIMSA will provide critical information to comprehend the crosstalk among various receptors on the plasma membrane.

Breast cancer progression results from subversion of multiple intra- or intercellular signaling pathways in normal mammary tissues and their microenvironment, which have an impact on cell differentiation, proliferation, migration, and angiogenesis. Phospholipases (PLC, PLD and PLA) are essential mediators of intraand intercellular signaling. They hydrolyze phospholipids, which are major components of cell membrane that can generate many bioactive lipid mediators, such as diacylglycerol, phosphatidic acid, lysophosphatidic acid, and arachidonic acid. Enzymatic processing of phospholipids by phospholipases converts these molecules into lipid mediators that regulate multiple cellular processes, which in turn can promote breast cancer progression. Thus, dysregulation of phospholipases contributes to a number of human diseases, including cancer. This review describes how phospholipases regulate multiple cancer-associated cellular processes, and the interplay among different phospholipases in breast cancer. A thorough understanding of the breast cancer-associated signaling networks of phospholipases is necessary to determine whether these enzymes are potential targets for innovative therapeutic strategies.

Fibrillar collagen and elastic fibers are the main components of the dermal extracellular matrix (ECM), which confers mechanical strength and resilience to the skin. In particular, type I collagen produced by fibroblasts is the most abundant collagen that determines the general strength of the ECM, thereby contributing to the prevesntion of the skin-aging process. Although the natural anthraquinone derivative emodin (1,3,8-trihydroxy-6-methylanthraquinone) exerts numerous beneficial effects, including antiviral, anticancer, anti-inflammatory and wound-healing effects in diverse cells, the effect of emodin on collagen expression or skin aging is not fully understood. The present study demonstrated that exposure to emodin increased type I collagen synthesis in a concentration- and time-dependent manner in Hs27 human dermal fibroblasts. Subsequent experiments showed that emodin strongly increased collagen type I levels without altering cell proliferation or cellular matrix metalloproteinase-1 (MMP-1) expression. Additionally, it was determined that increased phosphorylation of 5' AMP-activated protein kinase, following emodin treatment, was responsible for increased type I collagen synthesis. These findings clearly indicate that emodin plays an important role in collagen type I synthesis in dermal fibroblasts, thereby making it a potential drug candidate for treating skin aging and wrinkles.

Phospholipase D (PLD)2 via its enzymatic activity regulates cell proliferation and migration and thus is implicated in cancer. However, the role of PLD2 in obesity and type 2 diabetes has not previously been investigated. Here, we show that during diet-induced thermogenesis and obesity, levels of PLD2 but not PLD1 in adipose tissue are inversely related with uncoupling protein 1, a key thermogenic protein. We demonstrate that the thermogenic program in adipose tissue is significantly augmented in mice with adipocyte-specific Pld2 deletion or treated with a PLD2-specific inhibitor and that these mice are resistant to high fat diet–induced obesity, glucose intolerance, and insulin resistance. Mechanistically, we show that Pld2 deletion in adipose tissue or PLD2 pharmacoinhibition acts via p62 to improve mitochondrial quality and quantity in adipocytes. Thus, PLD2 inhibition is an attractive therapeutic approach for obesity and type 2 diabetes by resolving defects in diet-induced thermogenesis.

Aims. To investigate the association between parity and the risk of incident dementia in women. Methods. We pooled baseline and follow-up data for community-dwelling women aged 60 or older from six population-based, prospective cohort studies from four European and two Asian countries. We investigated the association between parity and incident dementia using Cox proportional hazards regression models adjusted for age, educational level, hypertension, diabetes mellitus and cohort, with additional analysis by dementia subtype (Alzheimer dementia (AD) and non-Alzheimer dementia (NAD)). Results. Of 9756 women dementia-free at baseline, 7010 completed one or more follow-up assessments. The mean follow-up duration was 5.4 ± 3.1 years and dementia developed in 550 participants. The number of parities was associated with the risk of incident dementia (hazard ratio (HR) = 1.07, 95% confidence interval (CI) = 1.02–1.13). Grand multiparity (five or more parities) increased the risk of dementia by 30% compared to 1–4 parities (HR = 1.30, 95% CI = 1.02–1.67). The risk of NAD increased by 12% for every parity (HR = 1.12, 95% CI = 1.02–1.23) and by 60% for grand multiparity (HR = 1.60, 95% CI = 1.00–2.55), but the risk of AD was not significantly associated with parity. Conclusions. Grand multiparity is a significant risk factor for dementia in women. This may have particularly important implications for women in low and middle-income countries where the fertility rate and prevalence of grand multiparity are high.

Metformin is the first-line therapy for type 2 diabetes, but there are large inter-individual variations in responses to this drug. Its mechanism of action is not fully understood, but activation of AMP-activated protein kinase (AMPK) and changes in the gut microbiota appear to be important. The inhibitory role of microbial metabolites on metformin action has not previously been investigated. Here, we show that concentrations of the microbial metabolite imidazole propionate are higher in subjects with type 2 diabetes taking metformin who have high blood glucose. We also show that metformin-induced glucose lowering is not observed in mice pretreated with imidazole propionate. Furthermore, we demonstrate that imidazole propionate inhibits AMPK activity by inducing inhibitory AMPK phosphorylation, which is dependent on imidazole propionate-induced basal Akt activation. Finally, we identify imidazole propionate-activated p38γ as a novel kinase for Akt and demonstrate that p38γ kinase activity mediates the inhibitory action of imidazole propionate on metformin.