Delineating the folding steps of helical-bundle membrane proteins has been a challenging task. Many questions remain unanswered, including the conformation and stability of the states populated ...during folding, the shape of the energy barriers between the states, and the role of lipids as a solvent in mediating the folding. Recently, theoretical frames have matured to a point that permits detailed dissection of the folding steps, and advances in experimental techniques at both single-molecule and ensemble levels enable selective modulation of specific steps for quantitative determination of the folding energy landscapes. We also discuss how lipid molecules would play an active role in shaping the folding energy landscape of membrane proteins, and how folding of multi-domain membrane proteins can be understood based on our current knowledge. We conclude this review by offering an outlook for emerging questions in the study of membrane protein folding.
Most cell signaling and surveillance circuits are physically maintained through a dense network of protein–protein interactions (PPIs). Genetic mutations, epigenetic changes as well as alterations in ...cellular microenvironment can markedly rewire the patterns of PPIs, which leads to neoplastic growth of cancer cells. There are accumulating evidences that drugs that target-specific PPI pairs may provide an opportunity to treat cancers with a higher specificity and efficacy than those inhibiting enzymatic activity of oncogenic proteins. Therefore, identification of driving PPIs in a given cancer not only improves our understanding for individual cancers, but it also provides therapeutic opportunities to cure the specific cancer. In this review, we introduce some examples of aberrant PPI complexes identified in several major types of cancers, and recent technical developments that permit assessment of PPI strength in clinical specimens. Finally, we discuss the potential use of such PPI profiling for the purpose of precision medicine.
Various mammalian cells, including cancer cells, shed extracellular vesicles (EVs), also known as exosomes and microvesicles, into surrounding tissues. These EVs play roles in tumor growth and ...metastasis by promoting angiogenesis. However, the detailed mechanism of how cancer-derived EVs elicit endothelial cell activation remains unknown. Here, we provide evidence that early growth response-1 (Egr-1) activation in endothelial cells is involved in the angiogenic activity of colorectal cancer cell-derived EVs. Both RNA interference-mediated downregulation of Egr-1 and ERK1/2 or JNK inhibitor significantly blocked EV-mediated Egr-1 activation and endothelial cell migration. Furthermore, lipid raft-mediated endocytosis inhibitor effectively blocked endothelial Egr-1 activation and migration induced by cancer-derived EVs. Our results suggest that Egr-1 activation in endothelial cells may be a key mechanism involved in the angiogenic activity of cancer-derived EVs. These findings will improve our understanding regarding the proangiogenic activities of EVs in diverse pathological conditions including cancer, cardiovascular diseases, and neurodegenerative diseases.
Purpose To investigate the incidence of corneal transplantation and identify rates and risk factors of repeated corneal transplantation in South Korea. Methods This is a retrospective ...population-based cohort study using the Korean National Health Insurance System database. Among the entire South Korean population (N = 51,827,813), we included those who underwent corneal transplantation more than once between January 2006 and December 2016, and analyzed the annual incidence of keratoplasty. The person-year incidence of repeated keratoplasty after the first operation was calculated according to risk factors including age group, sex, income level, surgical method, surgical etiology, and presence of major systemic diseases. Cox regression analysis was employed to evaluate the hazard ratios of those risk factors on repeated keratoplasty. Results A total of 9,452 cases of corneal transplantation occurred from January 2006 to December 2016. The average annual incidence of corneal transplantations was 1.694 per 100,000. The proportion of penetrating keratoplasty steadily decreased from 92.22% in 2006 to 77.81% in 2016. The average incidence of repeated keratoplasty among those who underwent corneal transplantation at least once was 43.24 per 1,000 person-years. Males had a greater incidence of repeated keratoplasty compared to females (males: 47.66 per 1,000, females: 36.04 per 1,000). The age group from 20 to 39 years demonstrated the lowest incidence of repeated keratoplasty at 24.94 per 1,000. Keratoconus had the lowest incidence of repeated keratoplasty (22.82 per 1,000). Conclusion This study may provide a better understanding of corneal diseases, help predict disease burden, and plan health care systems accordingly in South Korea.
Cancer cells actively release extracellular vesicles (EVs) as important carriers of cellular information to tumor microenvironments. Although the composition and quantity of the proteins contained in ...EVs are characterized, it remains unknown how these proteins in EVs are related to those in the original cells at the functional level. With epidermal growth factor receptor (EGFR) in lung adenocarcinoma cells as a model oncoprotein, it is studied how distinct types of EVs, microvesicles and exosomes, represent their original cells at the protein and protein–protein interaction (PPI) level. Using the recently developed single‐molecule immunolabeling and co‐immunoprecipitation schemes, the quantity and PPI strengths of EGFRs derived from EVs and the original lung adenocarcinoma cells are determined. It is found that the microvesicles exhibit higher correlations with the original cells than the exosomes in terms of the EGFR levels and their PPI patterns. In spite of these detailed differences between the microvesicles and exosomes, the EGFR PPI strengths measured for EVs generally show a tight correlation with those determined for the original cells. The results suggest that EGFRs contained in EVs closely reflect the cellular EGFR in terms of their downstream signaling capacity.
The quantity and protein–protein interaction (PPI) strengths of epidermal growth factor receptors (EGFRs) derived from extracellular vesicles (EVs) and the original lung adenocarcinoma cells are determined using single‐molecule immunolabeling and co‐immunoprecipitation methods. The EGFR PPI strengths measured for EVs generally show tight correlation with those determined for the original cells.
Hexagonal boron nitride (h-BN) and graphene have emerged as promising materials for proton exchange membranes because of their high proton conductivity and chemical stability. However, the defects ...and grain boundaries generated during the growth and transfer of two-dimensional materials limit their practical applicability. Here, we report the fabrication of membrane electrode assemblies using large-area single-oriented AA′-stacked trilayer h-BN (3L-BN), which exhibits very few defects during the growth and transfer, as a proton exchange membrane for use in fuel cell systems. The fuel cell based on AA′-stacked 3L-BN showed a H2 permeation current density as low as 2.69 mA cm–2 and an open circuit voltage (OCV) as high as 0.958 V; this performance is much superior to those for cells based on Nafion (3.7 mA cm–2 and 0.942 V, respectively) and single-layer h-BN (10.08 mA cm–2 and 0.894 V, respectively). Furthermore, the fuel cell with the AA′-stacked 3L-BN membrane almost maintained its original performance (OCV, maximum power density, and H2 permeation current density) even after 100 h of an accelerated stress test at 30% RH and 90 °C, while the fuel cells with the Nafion and single-layer BN membranes exhibited severely deteriorated performances. The stability of the cell based on the AA′-stacked 3L-BN membrane was better because the membrane prevented gas crossover and suppressed the generation of reactive radicals during cell operation.
To understand membrane protein biogenesis, we need to explore folding within a bilayer context. Here, we describe a single-molecule force microscopy technique that monitors the folding of helical ...membrane proteins in vesicle and bicelle environments. After completely unfolding the protein at high force, we lower the force to initiate folding while transmembrane helices are aligned in a zigzag manner within the bilayer, thereby imposing minimal constraints on folding. We used the approach to characterize the folding pathways of the
rhomboid protease GlpG and the human β
-adrenergic receptor. Despite their evolutionary distance, both proteins fold in a strict N-to-C-terminal fashion, accruing structures in units of helical hairpins. These common features suggest that integral helical membrane proteins have evolved to maximize their fitness with cotranslational folding.
The functionalized lipid shell of hybrid nanoparticles plays an important role for improving their biocompatibility and in vivo stability. Yet few efforts have been made to critically examine the ...shell structure of nanoparticles and its effect on cell–particle interaction. Here we develop a microfluidic chip allowing for the synthesis of structurally well-defined lipid-polymer nanoparticles of the same sizes, but covered with either lipid-monolayer-shell (MPs, monolayer nanoparticles) or lipid-bilayer-shell (BPs, bilayer nanoparticles). Atomic force microscope and atomistic simulations reveal that MPs have a lower flexibility than BPs, resulting in a more efficient cellular uptake and thus anticancer effect than BPs do. This flexibility-regulated cell–particle interaction may have important implications for designing drug nanocarriers.
DNA barcoding provides a way to label a myriad of different biological molecules using the extreme programmability in DNA sequence synthesis. Fluorescence imaging is presumably the most ...easy-to-access method for DNA barcoding, yet large spectral overlaps between fluorescence dyes severely limit the numbers of barcodes that can be detected simultaneously. We here demonstrate the use of single-molecule fluorescence resonance energy transfer (FRET) to encode virtual signals in DNA barcodes using conventional two-color fluorescence microscopy. By optimizing imaging and biochemistry conditions for weak DNA hybridization events, we markedly enhanced accuracy in our determination of the single-molecule FRET efficiency exhibited by each binding event between DNA barcode sequences. This allowed us to unambiguously differentiate six DNA barcodes encoding different FRET values without involving any probe sequence exchanges. Our method can be directly incorporated with previous DNA barcode techniques, and may thus be widely adopted to expand the signal space of DNA barcoding.
This study aims to enhance second language (L2) learners’ motivation and facilitate successful L2 learning by using motivational languaging, an intervention that encourages learners to reflect on, ...and externalize, their L2-speaking, competent future self-concepts by writing or speaking about them. Two types of effective languaging activities were developed: individual and group writing. Participants were 264 Grade 10 high school students, divided into three groups: one control group and two experimental groups. Using activity workbooks, the first experimental group engaged in ‘individual writing’ guided by a series of questions in their workbooks (e.g. reasons for learning English and their ideal images of using English). The second experimental group carried out ‘group writing’, including group discussions and writing of group members’ opinions. The activities were conducted in the participants’ first language, Korean, for 30–40 minutes once a week for six weeks. The results indicated, in the two experimental groups, that the participants’ L2 learning motivation was enhanced, including the ideal L2 self. When observing the changes in the influence of motivation on motivated L2 learning behavior and English proficiency, meaningful increases were only found in the individual writing group, with the ideal L2 self showing more powerful impact on the two criterion measures after the activities.
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