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Low-density lipoprotein receptor-related protein 6 (LRP6) is a coreceptor of the β-catenin-dependent Wnt signaling pathway. The LRP6 ectodomain binds Wnt proteins, as well as Wnt inhibitors such as sclerostin (SOST), which negatively regulates Wnt signaling in osteocytes. Although LRP6 ectodomain 1 (E1) is known to interact with SOST, several unresolved questions remain, such as the reason why SOST binds to LRP6 E1E2 with higher affinity than to the E1 domain alone. Here, we present the crystal structure of the LRP6 E1E2–SOST complex with two interaction sites in tandem. The unexpected additional binding site was identified between the C-terminus of SOST and the LRP6 E2 domain. This interaction was confirmed by in vitro binding and cell-based signaling assays. Its functional significance was further demonstrated in vivo using Xenopus laevis embryos. Our results provide insights into the inhibitory mechanism of SOST on Wnt signaling. The low-density lipoprotein receptor-related protein 6 (LRP6) is a co-receptor of the β-catenin-dependent Wnt signaling pathway and interacts with the Wnt inhibitor sclerostin (SOST). Here the authors present the crystal structure of SOST in complex with the LRP6 E1E2 ectodomain construct, which reveals that the SOST C-terminus binds to the LRP6 E2 domain, and further validate this binding site with in vitro and in vivo experiments.

Secretory proteins are an essential component of interorgan communication networks that regulate animal physiology. Current approaches for identifying secretory proteins from specific cell and tissue types are largely limited to in vitro or ex vivo models which often fail to recapitulate in vivo biology. As such, there is mounting interest in developing in vivo analytical tools that can provide accurate information on the origin, identity, and spatiotemporal dynamics of secretory proteins. Here, we describe i SLET (in situ Secretory protein Labeling via ER-anchored TurboID) which selectively labels proteins that transit through the classical secretory pathway via catalytic actions of Sec61b-TurboID, a proximity labeling enzyme anchored in the ER lumen. To validate i SLET in a whole-body system, we express i SLET in the mouse liver and demonstrate efficient labeling of liver secretory proteins which could be tracked and identified within circulating blood plasma. Furthermore, proteomic analysis of the labeled liver secretome enriched from liver i SLET mouse plasma is highly consistent with previous reports of liver secretory protein profiles. Taken together, i SLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets. The in vivo identification of proteins secreted from a specific cell type or tissue remains challenging. Here, the authors develop a proximity labeling-based method to selectively label secreted proteins and combine it with proteomics to identify liver secretory proteins in mouse plasma.

Recent advances in structural biology have profoundly enhanced our understanding of G protein-coupled receptors (GPCRs), providing detailed molecular insights into their activation and ligand recognition. Here, in this Review, we explore the molecular mechanisms of class A and class B GPCRs bound to peptide agonists and their implications for drug development. We examine representative GPCRs, such as the angiotensin II type 1 receptor, chemokine receptor 5, μ-opioid receptor, parathyroid hormone 1 receptor and glucagon-like peptide 1 receptor (GLP-1R), highlighting their activation processes upon peptide ligand binding. Comparative analysis of structures bound to endogenous and synthetic peptide ligands reveals critical insights for rational drug design. A case study on GLP-1R demonstrates how structural insights have led to the design of successful drugs for type 2 diabetes and obesity. This comparative structural analysis aims to deepen our understanding of GPCR activation mechanisms and support future drug discovery efforts targeting peptide-binding GPCRs. Peptide GPCRs reveal insights for targeted drug development G protein-coupled receptors (GPCRs) constitute a large family of membrane proteins that play a key role in transmitting signals from outside to inside the cell. Developing drugs that target certain GPCRs, especially those that bind peptide ligands, is challenging due to their structural complexity. Recent advances in X-ray crystallography and cryo-electron microscopy have enabled detailed structural analyses of these receptors. The study focuses on peptide-binding GPCRs involved in critical physiological processes such as metabolism, immune response and pain perception. By examining structures of GPCRs bound to various peptides and drugs, researchers revealed conformational changes associated with receptor activation and signal transduction. These insights enhance our understanding of ligand recognition and provide valuable guidance for designing more selective and effective therapeutics targeting peptide-binding GPCRs implicated in diseases such as diabetes and cardiovascular disorders. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

Anchoring components are added to wearable robots to ensure a stable interaction between the suits and the human body and to minimize the displacement of the suits. However, these components can apply pressure to the body and can cause user dissatisfaction, which can decrease willingness to use the suits. Therefore, this study aims to develop a suit-type soft-wearable robot platform for walking assistance by providing comfortable garment pressure to ensure user satisfaction. The first prototype of a wearable robot suit was developed with anchoring components on the shoulders, waist, and thighs based on previous research results. Wear tests were conducted to measure garment pressure depending on posture using pressure sensors, and satisfaction surveys were conducted. The second prototype design was then developed, and performance tests with flexible artificial muscles and a satisfaction survey were conducted. Regarding the first prototype, the participants felt more than normal pressure in the shoulders and relatively less pressure in the thighs and calves. Thus, compared to the first design, the second design ensured a decreased garment pressure and resulted in an improvement of overall user satisfaction. These results can help provide guidance in the development of wearable robots by taking pressure comfort and user satisfaction into consideration.

Small, compact genomes confer a selective advantage to viruses, yet human cytomegalovirus (HCMV) expresses the long non-coding RNAs (lncRNAs); RNA1.2, RNA2.7, RNA4.9, and RNA5.0. Little is known about the function of these lncRNAs in the virus life cycle. Here, we dissected the functional and molecular landscape of HCMV lncRNAs. We found that HCMV lncRNAs occupy ~ 30% and 50–60% of total and poly(A)+viral transcriptome, respectively, throughout virus life cycle. RNA1.2, RNA2.7, and RNA4.9, the three abundantly expressed lncRNAs, appear to be essential in all infection states. Among these three lncRNAs, depletion of RNA2.7 and RNA4.9 results in the greatest defect in maintaining latent reservoir and promoting lytic replication, respectively. Moreover, we delineated the global post-transcriptional nature of HCMV lncRNAs by nanopore direct RNA sequencing and interactome analysis. We revealed that the lncRNAs are modified with N 6 -methyladenosine (m 6 A) and interact with m 6 A readers in all infection states. In-depth analysis demonstrated that m 6 A machineries stabilize HCMV lncRNAs, which could account for the overwhelming abundance of viral lncRNAs. Our study lays the groundwork for understanding the viral lncRNA–mediated regulation of host-virus interaction throughout the HCMV life cycle.

Background Patients with estrogen receptor (ER)-positive breast cancer (BC) can be treated with endocrine therapy targeting ER, however, metastatic recurrence occurs in 25% of the patients who have initially been treated. Secreted proteins from tumors play important roles in cancer metastasis but previous methods for isolating secretory proteins had limitations in identifying novel targets. Methods We applied an in situ secretory protein labeling technique using TurboID to analyze secretome from tamoxifen-resistant (TAMR) BC. The increased expression of LGALS3BP was validated using western blotting, qPCR, ELISA, and IF. Chromatin immunoprecipitation was applied to analyze estrogen-dependent regulation of LGALS3BP transcription. The adhesive and angiogenic functions of LGALS3BP were evaluated by abrogating LGALS3BP expression using either shRNA-mediated knockdown or a neutralizing antibody. Xenograft mouse experiments were employed to assess the in vivo metastatic potential of TAMR cells and the LGALS3BP protein. Clinical evaluation of LGALS3BP risk was carried out with refractory clinical specimens from tamoxifen-treated ER-positive BC patients and publicly available databases. Results TAMR secretome analysis revealed that 176 proteins were secreted at least 2-fold more from MCF7/TAMR cells than from sensitive cells, and biological processes such as cell adhesion and angiogenesis were associated with the TAMR secretome. Galectin-3 binding protein (LGALS3BP) was one of the top 10 most highly secreted proteins in the TAMR secretome. The expression level of LGALS3BP was suppressed by estrogen signaling, which involves direct ERα binding to its promoter region. Secreted LGALS3BP in the TAMR secretome helped BC cells adhere to the extracellular matrix and promoted the tube formation of human umbilical vein endothelial cells. Compared with sensitive cells, xenograft animal experiments with MCF7/TAMR cells showed increased pulmonary metastasis, which completely disappeared in LGALS3BP-knockdown TAMR cells. Finally, higher levels of LGALS3BP were associated with poor prognosis in ER-positive BC patients treated with adjuvant tamoxifen in the clinic. Conclusion TAMR secretome analysis identified secretory proteins, such as LGALS3BP, that are involved in biological processes closely related to metastasis. Secreted LGALS3BP from the TAMR cells promoted adhesion of the cells to the extracellular matrix and vasculature formation, which may support metastasis of TAMR cells.

The CRISPR-Cas9 system has emerged as a powerful tool for precise genome editing, with ongoing research focused on enhancing its reliability and expanding its versatility. One effective strategy involves the integration of foreign functional domains into Cas9 to confer new capabilities. However, successful integration requires identification of insertion sites that preserve the protein’s structural integrity and function. In this study, we identified a C-terminal region of Streptococcus pyogenes Cas9 (SpCas9), spanning residues 1242–1263, as a viable site for domain replacement. Structural and biochemical analyses of a SpCas9 variant lacking this region confirmed its dispensability for SpCas9 activity. As a proof of concept, we substituted this segment with the evolved E. coli tRNA adenosine deaminase (TadA), a key component of adenine base editors. Functional evaluation of this engineered SpCas9-TadA variant demonstrated deamination efficiency comparable to that of the ABE8e, with the potential to modulate the editing window through linker design. These results highlight the potential of targeted engineering of this region to develop more precise and versatile genome editing tools.

CellNet is a computational platform designed to improve cell fate engineering protocols. Here the authors demonstrate how to apply CellNet to RNA-seq data and how to build a completely new CellNet platform applicable to new species or cell types/tissues. CellNet is a computational platform designed to assess cell populations engineered by either directed differentiation of pluripotent stem cells (PSCs) or direct conversion, and to suggest specific hypotheses to improve cell fate engineering protocols. CellNet takes as input gene expression data and compares them with large data sets of normal expression profiles compiled from public sources, in regard to the extent to which cell- and tissue-specific gene regulatory networks are established. CellNet was originally designed to work with human or mouse microarray expression data for 21 cell or tissue (C/T) types. Here we describe how to apply CellNet to RNA-seq data and how to build a completely new CellNet platform applicable to, for example, other species or additional cell and tissue types. Once the raw data have been preprocessed, running CellNet takes only several minutes, whereas the time required to create a completely new CellNet is several hours.

Quinoa is a pseudocereal that contains high quality protein, minerals, vitamins, polyphenols, and phytosterols. In this study, quinoa was fermented by Rhizopus oligosporus (R. oligosporus) up to 5 days and the functional compounds (l-carnitine, GABA, vanillic acid and gallic acid) were analyzed by LC/MS. The amounts of l-carnitine and GABA were 0.13 mg/kg and 540 mg/kg for nonfermented quinoa (NF), 3.15 mg/kg and 1040 mg/kg for fermented quinoa at 3 days (3F), and 1.54 mg/kg and 810 mg/kg for fermented quinoa at 5 days (5F). The vanillic acid and gallic acid were 1.3 and 0.1 mg/kg for NF, 1.55 and 2.37 mg/kg for 3F, and 1.83 and 0.84 mg/kg for 5F, respectively. Total phenolic contents and total flavonoids contents were 41 mg gallic acid (GAE)/kg and 13 mg quercetin equivalent (QE)/kg for NF, 74 mg GAE/kg and 16 mg QE/kg for 3F, and 80 mg GAE/kg and 19 mg QE/kg for 5F, respectively. Antioxidant activity (SC50) was 3.6 mg/mL for NF, 3.4 mg/mL for 3F, and 2.3 mg/mL for 5F. Nitric oxide production on RAW264.7 macrophages of fermented quinoa revealed 29% and 56% inhibition of nitric oxide production for NF and 5F, respectively. Therefore, fermented quinoa can be used as a healthy and valuable food product.