Mechanistic Insights into Inter-Organellar Membrane Contact Sites Based on Crystal Structures

Department of Biological SciencesEukaryotic cells contain various membrane-bound subcellular compartments called organelles. For cell survival, organelles perform specific functions respectively or cooperate through communication. Recently, as the development of electron microscopy, it has been discovered that organelles are located closely and make contact sites with about 10 nm distance between two membranes, which is called membrane contact sites (MCSs). MCSs are constructed and maintained stably or transiently by tethering structures mainly composed of proteins. It has been revealed that MCSs are important sites for various cellular functions such as intracellular signaling, lipid and ion exchange, organelle trafficking, and inheritance. Nucleus-vacuole junctions (NVJs) are one of the firstly identified MCSs in the budding yeast Saccharomyces cerevisiae. The formation of NVJs depends on the nuclear membrane protein Nvj1 and vacuolar protein Vac8. NVJs formed by the interaction between Vac8 and Nvj1 are critical for mediating piecemeal microautophagy of the nucleus (PMN), one of the selective microautophagy pathways in the yeast. Herein, we report the crystal structure of Vac8-Nvj1 complex. Vac8 comprises the H1 helix at the N terminus, followed by 12 armadillo repeats (ARMs). The 80 ??? extended loop of Nvj1 binds to the conserved inner groove of Vac8 ARM domain in an antiparallel manner. Disruption of the Vac8-Nvj1 interaction results in the loss of NVJ formation and further PMN in Saccharomyces cerevisiae. Vac8 cationic triad (Arg276, Arg317, and Arg359) motifs interacting with Nvj1 are also critical to the recognition of Atg13, a key component of the cytoplasm-to-vacuole targeting (CVT) pathway, indicating competitive binding to Vac8. Indeed, the crystal structure of Vac8-Atg13 complex reveals that the 70 ??? extended loop of Atg13 binds to the ARM domain of Vac8 in a similar manner with Nvj1. The interaction between Vac8 and Atg13 is also essential for mediating CVT pathway in Saccharomyces cerevisiae. Structural, biochemical, and in vivo experiments demonstrate that the H1 helix of Vac8 intramolecularly interacts with the first ARM and regulates its self-association, which is crucial for PMN and CVT pathway. Crystal structures also reveal that Vac8-Nvj1 and Vac8-Atg13 form heterotetramer with the different quaternary organization. The structural comparison provides a molecular understanding of how a single ARM domain protein adopts different quaternary structures depending on its binding proteins to differentially regulate two closely related but distinct cellular pathways. Next, recent research suggested that 3???-5??? exonuclease domain-containing protein 2, EXD2 plays an essential role as a new component of DNA double-strand break repair machinery in the nucleus with a conserved DEDDy superfamily 3???-5??? exonuclease domain. However, the following researches raised the possibility that EXD2 might be localized to mitochondria with unclear mitochondrial sublocation. Herein, electron microscope imaging analysis and proximity labeling reveals that EXD2 is anchored to the mitochondrial outer membrane through a conserved N-terminal transmembrane domain, while the C-terminal region is cytosolic. The crystal structure of the exonuclease domain reveals a domain-swapped dimer in which the central ??5????7 helices are mutually crossed over, resulting in chimeric active sites. Additionally, the C-terminal segments absent in other DnaQ family exonucleases enclose the central chimeric active sites. Structural analyses of crystal structures in complex with Mn2+/Mg2+ and biochemical experiments demonstrate that unusual dimeric organization and additional C-segments stabilize the active site, facilitate discrimination between DNA and RNA substrates based on divalent cation coordination, and generate a positively charged groove that binds substrates. Based on structural studies and previously reported data, we suggest that EXD2 can play possible roles in the mitochondrial outer membrane or inter-mitochondrial membrane contact sites with its novel exonuclease activity.ope

Quaternary structures of Vac8 differentially regulate the Cvt and PMN pathways.

Armadillo (ARM) repeat proteins constitute a large protein family with diverse and fundamental functions in all organisms, and armadillo repeat domains share high structural similarity. However, exactly how these structurally similar proteins can mediate diverse functions remains a long-standing question. Vac8 (vacuole related 8) is a multifunctional protein that plays pivotal roles in various autophagic pathways, including piecemeal microautophagy of the nucleus (PMN) and cytoplasm-to-vacuole targeting (Cvt) pathways in the budding yeast Saccharomyces cerevisiae. Vac8 comprises an H1 helix at the N terminus, followed by 12 armadillo repeats. Herein, we report the crystal structure of Vac8 bound to Atg13, a key component of autophagic machinery. The 70-angstrom extended loop of Atg13 binds to the ARM domain of Vac8 in an antiparallel manner. Structural, biochemical, and in vivo experiments demonstrated that the H1 helix of Vac8 intramolecularly associates with the first ARM and regulates its self-association, which is crucial for Cvt and PMN pathways. The structure of H1 helix-deleted Vac8 complexed with Atg13 reveals that Vac8[Delta 19-33]-Atg13 forms a heterotetramer and adopts an extended superhelical structure exclusively employed in the Cvt pathway. Most importantly, comparison of Vac8-Nvj1 and Vac8-Atg13 provides a molecular understanding of how a single ARM domain protein adopts different quaternary structures depending on its associated proteins to differentially regulate 2 closely related but distinct cellular pathways

Peptidyl-prolyl cis/trans isomerase Pin1 interacts with hepatitis B virus core particle, but not with HBc protein, to promote HBV replication

Here, we demonstrate that the peptidyl-prolyl cis/trans isomerase Pin1 interacts noncovalently with the hepatitis B virus (HBV) core particle through phosphorylated serine/threonine-proline (pS/TP) motifs in the carboxyl-terminal domain (CTD) but not with particle-defective, dimer-positive mutants of HBc. This suggests that neither dimers nor monomers of HBc are Pin1-binding partners. The 162TP, 164SP, and 172SP motifs within the HBc CTD are important for the Pin1/core particle interaction. Although Pin1 dissociated from core particle upon heat treatment, it was detected as an opened-up core particle, demonstrating that Pin1 binds both to the outside and the inside of the core particle. Although the amino-terminal domain S/TP motifs of HBc are not involved in the interaction, 49SP contributes to core particle stability, and 128TP might be involved in core particle assembly, as shown by the decreased core particle level of S49A mutant through repeated freeze and thaw and low-level assembly of the T128A mutant, respectively. Overexpression of Pin1 increased core particle stability through their interactions, HBV DNA synthesis, and virion secretion without concomitant increases in HBV RNA levels, indicating that Pin1 may be involved in core particle assembly and maturation, thereby promoting the later stages of the HBV life cycle. By contrast, parvulin inhibitors and PIN1 knockdown reduced HBV replication. Since more Pin1 proteins bound to immature core particles than to mature core particles, the interaction appears to depend on the stage of virus replication. Taken together, the data suggest that physical association between Pin1 and phosphorylated core particles may induce structural alterations through isomerization by Pin1, induce dephosphorylation by unidentified host phosphatases, and promote completion of virus life cycle

The structure of human EXD2 reveals a chimeric 3' to 5' exonuclease domain that discriminates substrates via metal coordination.

EXD2 (3'-5' exonuclease domain-containing protein 2) is an essential protein with a conserved DEDDy superfamily 3'-5' exonuclease domain. Recent research suggests that EXD2 has two potential functions: as a component of the DNA double-strand break repair machinery and as a ribonuclease for the regulation of mitochondrial translation. Herein, electron microscope imaging analysis and proximity labeling revealed that EXD2 is anchored to the mitochondrial outer membrane through a conserved N-terminal transmembrane domain, while the C-terminal region is cytosolic. Crystal structures of the exonuclease domain in complex with Mn2+/Mg2+ revealed a domain-swapped dimer in which the central α5-α7 helices are mutually crossed over, resulting in chimeric active sites. Additionally, the C-terminal segments absent in other DnaQ family exonucleases enclose the central chimeric active sites. Combined structural and biochemical analyses demonstrated that the unusual dimeric organization stabilizes the active site, facilitates discrimination between DNA and RNA substrates based on divalent cation coordination and generates a positively charged groove that binds substrates.Cell Logistics Research Center [2016R1A5A1007318]; Basic Research Program, National Research Foundation of Korea [NRF-2019R1A2C3008463]; Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea [HI18C1395]; Institute for Basic Science [IBS-R022-D1]. Funding for open access charge: Cell Logistics Research Center, National Research Foundation of Korea [2016R1A5A1007318]

Charge Injection and Energy Transfer of Surface-Engineered InP/ZnSe/ZnS Quantum Dots

Surface passivation is a critical aspect of preventing surface oxidation and improving the emission properties of nanocrystal quantum dots (QDs). Recent studies have demonstrated the critical role of surface ligands in determining the performance of QD-based light-emitting diodes (QD-LEDs). Herein, the underlying mechanism by which the capping ligands of InP/ZnSe/ZnS QDs influence the brightness and lifetime of the QD-LEDs is investigated. The electrochemical results demonstrate that highly luminescent InP/ZnSe/ZnS QDs exhibit modulated charge injection depending on the length of the surface ligand chains: short alkyl chains on the ligands are favorable for charge transport to the QDs. In addition, the correlation between the spectroscopic and XRD analyses suggests that the length of the ligand chain tunes the ligand–ligand coupling strength, thereby controlling the inter-QD energy transfer dynamics. The present findings shed new light on the crucial role of surface ligands for InP/ZnSe/ZnS QD-LED applications

Virtual reality education program for women with uterine tumors treated by high-intensity focused ultrasound

This study aimed to develop and determine the effects of a nursing education program using virtual reality (VR) for women with uterine tumors undergoing treatment with high-intensity focused ultrasound (HIFU). Various nursing education methods need to be developed alongside new treatment methods and their effects should be clinically verified. Nursing intervention using VR has recently been attempted. The study comprises a pre- and post-test design with a non-equivalent control group.We assigned 54 women to experimental (n = 26) and control (n = 28) groups. The patients were diagnosed with benign uterine tumors and were treated with HIFU at two women's hospitals in D city. Data collected from these hospitals were analyzed using descriptive statistics, a pre-test of homogeneity, independent t-tests, and repeated measures analysis of variance. In the experimental group, uncertainty (t = 4.26, p < 0.001) and anxiety (t = 4.09, p < 0.001) were significantly lower compared to the control group. However, nursing satisfaction was significantly higher in the experimental group than in the control group (t = −4.50, p < 0.001). The VR education program is an educational nursing intervention that reduces uncertainty and anxiety and improves nursing satisfaction among women with uterine tumors treated by HIFU. We suggest that future nursing research integrates and converges disciplines according to progressive treatment methods and technological advancements for patients

Deep Learning Based Detection of Missing Tooth Regions for Dental Implant Planning in Panoramic Radiographic Images

Dental implantation is a surgical procedure in oral and maxillofacial surgery. Detecting missing tooth regions is essential for planning dental implant placement. This study proposes an automated method that detects regions of missing teeth in panoramic radiographic images. Tooth instance segmentation is required to accurately detect a missing tooth region in panoramic radiographic images containing obstacles, such as dental appliances or restoration. Therefore, we constructed a dataset that contains 455 panoramic radiographic images and annotations for tooth instance segmentation and missing tooth region detection. First, the segmentation model segments teeth into the panoramic radiographic image and generates teeth masks. Second, a detection model uses the teeth masks as input to predict regions of missing teeth. Finally, the detection model identifies the position and number of missing teeth in the panoramic radiographic image. We achieved 92.14% mean Average Precision (mAP) for tooth instance segmentation and 59.09% mAP for missing tooth regions detection. As a result, this method assists diagnosis by clinicians to detect missing teeth regions for implant placement

Deep Learning Based Detection of Missing Tooth Regions for Dental Implant Planning in Panoramic Radiographic Images

Dental implantation is a surgical procedure in oral and maxillofacial surgery. Detecting missing tooth regions is essential for planning dental implant placement. This study proposes an automated method that detects regions of missing teeth in panoramic radiographic images. Tooth instance segmentation is required to accurately detect a missing tooth region in panoramic radiographic images containing obstacles, such as dental appliances or restoration. Therefore, we constructed a dataset that contains 455 panoramic radiographic images and annotations for tooth instance segmentation and missing tooth region detection. First, the segmentation model segments teeth into the panoramic radiographic image and generates teeth masks. Second, a detection model uses the teeth masks as input to predict regions of missing teeth. Finally, the detection model identifies the position and number of missing teeth in the panoramic radiographic image. We achieved 92.14% mean Average Precision (mAP) for tooth instance segmentation and 59.09% mAP for missing tooth regions detection. As a result, this method assists diagnosis by clinicians to detect missing teeth regions for implant placement