A Critical Role for ERO1α in Arterial Thrombosis and Ischemic Stroke.

BACKGROUND: Platelet adhesion and aggregation play a crucial role in arterial thrombosis and ischemic stroke. Here, we identify platelet ERO1α (endoplasmic reticulum oxidoreductase 1α) as a novel regulator of Ca2+ signaling and a potential pharmacological target for treating thrombotic diseases. METHODS: Intravital microscopy, animal disease models, and a wide range of cell biological studies were utilized to demonstrate the pathophysiological role of ERO1α in arteriolar and arterial thrombosis and to prove the importance of platelet ERO1α in platelet activation and aggregation. Mass spectrometry, electron microscopy, and biochemical studies were used to investigate the molecular mechanism. We used novel blocking antibodies and small-molecule inhibitors to study whether ERO1α can be targeted to attenuate thrombotic conditions. RESULTS: Megakaryocyte-specific or global deletion of Ero1α in mice similarly reduced platelet thrombus formation in arteriolar and arterial thrombosis without affecting tail bleeding times and blood loss following vascular injury. We observed that platelet ERO1α localized exclusively in the dense tubular system and promoted Ca2+ mobilization, platelet activation, and aggregation. Platelet ERO1α directly interacted with STIM1 (stromal interaction molecule 1) and SERCA2 (sarco/endoplasmic reticulum Ca2+-ATPase 2) and regulated their functions. Such interactions were impaired in mutant STIM1-Cys49/56Ser and mutant SERCA2-Cys875/887Ser. We found that ERO1α modified an allosteric Cys49-Cys56 disulfide bond in STIM1 and a Cys875-Cys887 disulfide bond in SERCA2, contributing to Ca2+ store content and increasing cytosolic Ca2+ levels during platelet activation. Inhibition of Ero1α with small-molecule inhibitors but not blocking antibodies attenuated arteriolar and arterial thrombosis and reduced infarct volume following focal brain ischemia in mice. CONCLUSIONS: Our results suggest that ERO1α acts as a thiol oxidase for Ca2+ signaling molecules, STIM1 and SERCA2, and enhances cytosolic Ca2+ levels, promoting platelet activation and aggregation. Our study provides evidence that ERO1α may be a potential target to reduce thrombotic events.

Analysis of Changes in Sleep Quality and Patterns after Hip Fracture Using Real Evidence of Artificial Intelligence Linked (REAL) Hip Cohort Data.

Background and Objectives: Hip fractures are commonly found in elderly patients, and often result in chronic pain and decreased physical function, as well as worsening of overall health. It is known that early surgical intervention during the acute phase and rehabilitation are important for improving clinical outcomes for these patients. However, the importance of management for improving the quality of life of these patients is becoming more emphasized. Studies on changes in sleep patterns after hip fractures are rare overseas. Therefore, the aim of this study is to investigate the prevalence of sleep disturbance in patients with hip fractures and to analyze the changes in sleep disturbance after surgery by comparing the preoperative and postoperative results. Materials and Methods: During the period from August 2022 to January 2023, patients who underwent surgical treatment for hip fractures and were recruited into the REAL Hip Cohort were selected as research subjects. The sleep survey was conducted using the Pittsburgh Sleep Quality Index (PSQI). The PSQI is composed of 18 questions, each divided into areas of sleep quality, sleep latency, duration, efficiency, disturbance, use of medication, and daytime dysfunction. Each area is scored 0-3 points and the total is 0-21. A score greater than five indicates sleep disorder. The PSQI was surveyed during hospitalization and three months after surgery for post-fracture sleep status. To analyze changes before and after the fracture, paired T-tests and chi-square tests were performed. Results: From August 2022 to January 2023, a total of 40 patients who were recruited into the REAL Hip Cohort responded to the PSQI survey. The average age was 77.4 years and 36 were female. Sleep quality worsened from 0.75 ± 1.0 before surgery to 1.4 ± 1.0 three months after surgery (p = 0.019), and sleep efficiency also worsened from 0.4 ± 0.6 to 1.4 ± 1.0 (p < 0.001). The PSQI increased from an average of 5.2 ± 2.8 before surgery to 8.2 ± 4.2 three months after surgery (p = 0.007), and the number of patients who could be diagnosed with sleep disorders also increased from 12 (40%) to 24 (60%) (p = 0.030). Conclusions: A decline in overall sleep status was observed in patients in a survey on sleep patterns three months after hip fracture. Additional management is needed to improve their sleep patterns.

Longitudinal intravital imaging of cerebral microinfarction reveals a dynamic astrocyte reaction leading to glial scar formation.

Cerebral microinfarct increases the risk of dementia. But how microscopic cerebrovascular disruption affects the brain tissue in cellular-level are mostly unknown. Herein, with a longitudinal intravital imaging, we serially visualized in vivo dynamic cellular-level changes in astrocyte, pericyte and neuron as well as microvascular integrity after the induction of cerebral microinfarction for 1 month in mice. At day 2-3, it revealed a localized edema with acute astrocyte loss, neuronal death, impaired pericyte-vessel coverage and extravascular leakage of 3 kDa dextran (but not 2 MDa dextran) indicating microinfarction-related blood-brain barrier (BBB) dysfunction for small molecules. At day 5, the local edema disappeared with the partial restoration of microcirculation and recovery of pericyte-vessel coverage and BBB integrity. But brain tissue continued to shrink with persisted loss of astrocyte and neuron in microinfarct until 30 days, resulting in a collagen-rich fibrous scar surrounding the microinfarct. Notably, reactive astrocytes expressing glial fibrillary acidic protein (GFAP) appeared at the peri-infarct area early at day 2 and thereafter accumulated in the peri-infarct until 30 days, inducing glial scar formation in cerebral cortex. Our longitudinal intravital imaging of serial microscopic neurovascular pathophysiology in cerebral microinfarction newly revealed that astrocytes are critically susceptible to the acute microinfarction and their reactive response leads to the fibrous glial scar formation.

Dll4 Suppresses Transcytosis for Arterial Blood-Retinal Barrier Homeostasis.

RATIONALE: Central nervous system has low vascular permeability by organizing tight junction (TJ) and limiting endothelial transcytosis. While TJ has long been considered to be responsible for vascular barrier in central nervous system, suppressed transcytosis in endothelial cells is now emerging as a complementary mechanism. Whether transcytosis regulation is independent of TJ and its dysregulation dominantly causes diseases associated with edema remain elusive. Dll4 signaling is important for various vascular contexts, but its role in the maintenance of vascular barrier in central nervous system remains unknown. OBJECTIVE: To find a TJ-independent regulatory mechanism selective for transcytosis and identify its dysregulation as a cause of pathological leakage. METHODS AND RESULTS: We studied transcytosis in the adult mouse retina with low vascular permeability and employed a hypertension-induced retinal edema model for its pathological implication. Both antibody-based and genetic inactivation of Dll4 or Notch1 induce hyperpermeability by increasing transcytosis without junctional destabilization in arterial endothelial cells, leading to nonhemorrhagic leakage predominantly in the superficial retinal layer. Endothelial Sox17 deletion represses Dll4 in retinal arteries, phenocopying Dll4 blocking-driven vascular leakage. Ang II (angiotensin II)-induced hypertension represses arterial Sox17 and Dll4, followed by transcytosis-driven retinal edema, which is rescued by a gain of Notch activity. Transcriptomic profiling of retinal endothelial cells suggests that Dll4 blocking activates SREBP1 (sterol regulatory element-binding protein 1)-mediated lipogenic transcription and enriches gene sets favorable for caveolae formation. Profiling also predicts the activation of VEGF (vascular endothelial growth factor) signaling by Dll4 blockade. Inhibition of SREBP1 or VEGF-VEGFR2 (VEGF receptor 2) signaling attenuates both Dll4 blockade-driven and hypertension-induced retinal leakage. CONCLUSIONS: In the retina, Sox17-Dll4-SREBP1 signaling axis controls transcytosis independently of TJ in superficial arteries among heterogeneous regulations for the whole vessels. Uncontrolled transcytosis via dysregulated Dll4 underlies pathological leakage in hypertensive retina and could be a therapeutic target for treating hypertension-associated retinal edema.

χ-separation: Magnetic susceptibility source separation toward iron and myelin mapping in the brain.

Obtaining a histological fingerprint from the in-vivo brain has been a long-standing target of magnetic resonance imaging (MRI). In particular, non-invasive imaging of iron and myelin, which are involved in normal brain functions and are histopathological hallmarks in neurodegenerative diseases, has practical utilities in neuroscience and medicine. Here, we propose a biophysical model that describes the individual contribution of paramagnetic (e.g., iron) and diamagnetic (e.g., myelin) susceptibility sources to the frequency shift and transverse relaxation of MRI signals. Using this model, we develop a method, χ-separation, that generates the voxel-wise distributions of the two sources. The method is validated using computer simulation and phantom experiments, and applied to ex-vivo and in-vivo brains. The results delineate the well-known histological features of iron and myelin in the specimen, healthy volunteers, and multiple sclerosis patients. This new technology may serve as a practical tool for exploring the microstructural information of the brain.

A geometric approach to separate the effects of magnetic susceptibility and chemical shift/exchange in a phantom with isotropic magnetic susceptibility.

PURPOSE: To separate the effects of magnetic susceptibility and chemical shift/exchange in a phantom with isotropic magnetic susceptibility, and to generate a chemical shift/exchange-corrected QSM result. METHODS: Magnetic susceptibility and chemical shift/exchange are the properties of a material. Both are known to induce the resonance frequency shift in MRI. In current QSM, the susceptibility is reconstructed from the frequency shift, ignoring the contribution of the chemical shift/exchange. In this work, a simple geometric approach, which averages the frequency shift maps from three orthogonal B0 directions to generate a chemical shift/exchange map, is developed using the fact that the average nullifies the (isotropic) susceptibility effects. The resulting chemical shift/exchange map is subtracted from the total frequency shift, producing a frequency shift map solely from susceptibility. Finally, this frequency shift map is reconstructed to a susceptibility map using a QSM algorithm. The proposed method is validated in numerical simulations and applied to phantom experiments with olive oil, bovine serum albumin, ferritin, and iron oxide solutions. RESULTS: Both simulations and experiments confirm that the method successfully separates the contributions of the susceptibility and chemical shift/exchange, reporting the susceptibility and chemical shift/exchange of olive oil (susceptibility: 0.62 ppm, chemical shift: -3.60 ppm), bovine serum albumin (susceptibility: -0.059 ppm, chemical shift: 0.008 ppm), ferritin (susceptibility: 0.125 ppm, chemical shift: -0.005 ppm), and iron oxide (susceptibility: 0.30 ppm, chemical shift: -0.039 ppm) solutions. CONCLUSION: The proposed method successfully separates the susceptibility and chemical shift/exchange in phantoms with isotropic magnetic susceptibility.

3D Visualization of Dynamic Cellular Reaction of Pulpal CD11c+ Dendritic Cells against Pulpitis in Whole Murine Tooth.

In dental pulp, diverse types of cells mediate the dental pulp immunity in a highly complex and dynamic manner. Yet, 3D spatiotemporal changes of various pulpal immune cells dynamically reacting against foreign pathogens during immune response have not been well characterized. It is partly due to the technical difficulty in detailed 3D comprehensive cellular-level observation of dental pulp in whole intact tooth beyond the conventional histological analysis using thin tooth slices. In this work, we validated the optical clearing technique based on modified Murray's clear as a valuable tool for a comprehensive cellular-level analysis of dental pulp. Utilizing the optical clearing, we successfully achieved a 3D visualization of CD11c+ dendritic cells in the dentin-pulp complex of a whole intact murine tooth. Notably, a small population of unique CD11c+ dendritic cells extending long cytoplasmic processes into the dentinal tubule while located at the dentin-pulp interface like odontoblasts were clearly visualized. 3D visualization of whole murine tooth enabled a reliable observation of these rarely existing cells with a total number less than a couple of tens in one tooth. These CD11c+ dendritic cells with processes in the dentinal tubule were significantly increased in the dental pulpitis model induced by mechanical and chemical irritation. Additionally, the 3D visualization revealed a distinct spatial 3D arrangement of pulpal CD11c+ cells in the pulp into a front-line barrier-like formation in the pulp within 12 h after the irritation. Collectively, these observations demonstrated the unique capability of optical clearing-based comprehensive 3D cellular-level visualization of the whole tooth as an efficient method to analyze 3D spatiotemporal changes of various pulpal cells in normal and pathological conditions.

SSD-TSEFFM: New SSD Using Trident Feature and Squeeze and Extraction Feature Fusion.

The single shot multi-box detector (SSD) exhibits low accuracy in small-object detection; this is because it does not consider the scale contextual information between its layers, and the shallow layers lack adequate semantic information. To improve the accuracy of the original SSD, this paper proposes a new single shot multi-box detector using trident feature and squeeze and extraction feature fusion (SSD-TSEFFM); this detector employs the trident network and the squeeze and excitation feature fusion module. Furthermore, a trident feature module (TFM) is developed, inspired by the trident network, to consider the scale contextual information. The use of this module makes the proposed model robust to scale changes owing to the application of dilated convolution. Further, the squeeze and excitation block feature fusion module (SEFFM) is used to provide more semantic information to the model. The SSD-TSEFFM is compared with the faster regions with convolution neural network features (RCNN) (2015), SSD (2016), and DF-SSD (2020) on the PASCAL VOC 2007 and 2012 datasets. The experimental results demonstrate the high accuracy of the proposed model in small-object detection, in addition to a good overall accuracy. The SSD-TSEFFM achieved 80.4% mAP and 80.2% mAP on the 2007 and 2012 datasets, respectively. This indicates an average improvement of approximately 2% over other models.

Improvement of reproducibility in quantitative susceptibility mapping (QSM) and transverse relaxation rates ( R2* ) after physiological noise correction.

BACKGROUND: Numerous studies have suggested that quantitative susceptibility mapping (QSM) and transverse relaxation rates ( R2* ) are useful to monitor neurological diseases. For clinical use of QSM and R2* , reproducibility is an important feature. However, respiration-induced local magnetic field variation makes artifacts in gradient echo-based images and reduces the reproducibility of QSM and R2* . PURPOSE: To investigate the improvement of reproducibility of QSM and R2* after the correction of respiration-induced field variation, and to assess the effect of varying types of the region of interest (ROI) analysis on reproducibility. STUDY TYPE: Reproducibility study. POPULATION: Ten controls. FIELD STRENGTH/SEQUENCE: 3T/multiecho gradient echo sequence. ASSESSMENT: Intrascan reproducibility of QSM and R2* was investigated in ROIs before and after the respiration correction. STATISTICAL TESTS: Reproducibility was obtained by the square of voxel-wise correlation coefficients between scans. A paired t-test was performed for comparison between before and after the respiration correction and between QSM and R2* . RESULTS: Based on the ROI analysis, reproducibility increased after the respiration correction. Reproducibility in the white matter (11.89% increased in QSM and 23.38% in R2* , P = 0.009 and 0.024, respectively) and deep gray matter (5.50% increased in QSM and 13.96% in R2* , P = 0.024 and 0.019, respectively) increased significantly after the respiration correction. Reproducibility of R2* was higher than that of QSM in the whole brain and cortical gray matter, while QSM maps showed higher reproducibility than R2* in the white matter and deep gray matter. DATA CONCLUSION: Respiration-induced error correction significantly improved reproducibility in QSM and R2* mapping. QSM and R2* mapping showed a different level of reproducibility depending on the types of ROI analysis. LEVEL OF EVIDENCE: 4 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2018.

Energy-Efficient Cluster-Head Selection for Wireless Sensor Networks Using Sampling-Based Spider Monkey Optimization.

Extending the lifetime and stability of wireless sensor networks (WSNs) through efficient energy consumption remains challenging. Though clustering has improved energy efficiency through cluster-head selection, its application is still complicated. In existing cluster-head selection methods, the locations where cluster-heads are desirable are first searched. Next, the nodes closest to these locations are selected as the cluster-heads. This location-based approach causes problems such as increased computation, poor selection accuracy, and the selection of duplicate nodes. To solve these problems, we propose the sampling-based spider monkey optimization (SMO) method. If the sampling population consists of nodes to select cluster-heads, the cluster-heads are selected among the nodes. Thus, the problems caused by different locations of nodes and cluster-heads are resolved. Consequently, we improve lifetime and stability of WSNs through sampling-based spider monkey optimization and energy-efficient cluster head selection (SSMOECHS). This study describes how the sampling method is used in basic SMO and how to select cluster-heads using sampling-based SMO. The experimental results are compared to similar protocols, namely low-energy adaptive clustering hierarchy centralized (LEACH-C), particle swarm optimization clustering protocol (PSO-C), and SMO based threshold-sensitive energy-efficient delay-aware routing protocol (SMOTECP), and the results are shown in both homogeneous and heterogeneous setups. In these setups, SSMOECHS improves network lifetime and stability periods by averages of 13.4%, 7.1%, 34.6%, and 1.8%, respectively.

Dilated Skip Convolution for Facial Landmark Detection.

Facial landmark detection has gained enormous interest for face-related applications due to its success in facial analysis tasks such as facial recognition, cartoon generation, face tracking and facial expression analysis. Many studies have been proposed and implemented to deal with the challenging problems of localizing facial landmarks from given images, including large appearance variations and partial occlusion. Studies have differed in the way they use the facial appearances and shape information of input images. In our work, we consider facial information within both global and local contexts. We aim to obtain local pixel-level accuracy for local-context information in the first stage and integrate this with knowledge of spatial relationships between each key point in a whole image for global-context information in the second stage. Thus, the pipeline of our architecture consists of two main components: (1) a deep network for local-context subnet that generates detection heatmaps via fully convolutional DenseNets with additional kernel convolution filters and (2) a dilated skip convolution subnet-a combination of dilated convolutions and skip-connections networks-that are in charge of robustly refining the local appearance heatmaps. Through this proposed architecture, we demonstrate that our approach achieves state-of-the-art performance on challenging datasets-including LFPW, HELEN, 300W and AFLW2000-3D-by leveraging fully convolutional DenseNets, skip-connections and dilated convolution architecture without further post-processing.

Whole brain g-ratio mapping using myelin water imaging (MWI) and neurite orientation dispersion and density imaging (NODDI).

MR g-ratio, which measures the ratio of the aggregate volume of axons to that of fibers in a voxel, is a potential biomarker for white matter microstructures. In this study, a new approach for acquiring an in-vivo whole human brain g-ratio map is proposed. To estimate the g-ratio, myelin volume fraction and axonal volume fraction are acquired using multi-echo gradient echo myelin water imaging (GRE-MWI) and neurite orientation dispersion and density imaging (NODDI), respectively. In order to translate myelin water fraction measured in GRE-MWI into myelin volume fraction, a new scaling procedure is proposed and validated. This scaling approach utilizes geometric measures of myelin structure and, therefore, provides robustness over previous methods. The resulting g-ratio map reveals an expected range of g-ratios (0.71-0.85 in major fiber bundles) with a small inter-subject coefficient of variance (less than 2%). Additionally, a few fiber bundles (e.g. cortico-spinal tract and optic radiation) show different constituents of myelin volume fraction and axonal volume fraction, indicating potentials to utilize the measures for deciphering fiber tracking.

Quantitative susceptibility mapping using deep neural network: QSMnet.

Deep neural networks have demonstrated promising potential for the field of medical image reconstruction, successfully generating high quality images for CT, PET and MRI. In this work, an MRI reconstruction algorithm, which is referred to as quantitative susceptibility mapping (QSM), has been developed using a deep neural network in order to perform dipole deconvolution, which restores magnetic susceptibility source from an MRI field map. Previous approaches of QSM require multiple orientation data (e.g. Calculation of Susceptibility through Multiple Orientation Sampling or COSMOS) or regularization terms (e.g. Truncated K-space Division or TKD; Morphology Enabled Dipole Inversion or MEDI) to solve an ill-conditioned dipole deconvolution problem. Unfortunately, they either entail challenges in data acquisition (i.e. long scan time and multiple head orientations) or suffer from image artifacts. To overcome these shortcomings, a deep neural network, which is referred to as QSMnet, is constructed to generate a high quality susceptibility source map from single orientation data. The network has a modified U-net structure and is trained using COSMOS QSM maps, which are considered as gold standard. Five head orientation datasets from five subjects were employed for patch-wise network training after doubling the training data using a model-based data augmentation. Seven additional datasets of five head orientation images (i.e. total 35 images) were used for validation (one dataset) and test (six datasets). The QSMnet maps of the test dataset were compared with the maps from TKD and MEDI for their image quality and consistency with respect to multiple head orientations. Quantitative and qualitative image quality comparisons demonstrate that the QSMnet results have superior image quality to those of TKD or MEDI results and have comparable image quality to those of COSMOS. Additionally, QSMnet maps reveal substantially better consistency across the multiple head orientation data than those from TKD or MEDI. As a preliminary application, the network was further tested for three patients, one with microbleed, another with multiple sclerosis lesions, and the third with hemorrhage. The QSMnet maps showed similar lesion contrasts with those from MEDI, demonstrating potential for future applications.

Comprehensive somatic genome alterations of urachal carcinoma.

BACKGROUND: Urachal cancer is a rare cancer that develops in the urachus. Because of its rarity, standard treatment therapies for urachal cancer are not established, and chemotherapeutic regimens for bladder cancer have been unsuccessful for patients with urachal cancer. Hence, we aim to understand a systematic molecular characterisation of urachal cancer. METHODS: We identified somatic single-nucleotide variations (SNVs)/indels and somatic copy number aberrations (SCNAs) in the 17 patients by using whole-exome sequencing (WES) and OncoScan platform (Affymetrix) as follows: tumour-normal paired sequencing (WES, n=10), tumour-only sequencing (WES, n=1; targeted deep sequencing, n=16), and OncoScan (n=17). RESULTS: Our analyses identified 27 genes with somatic SNVs and indels, as well as six genes (APC, COL5A1, KIF26B, LRP1B, SMAD4 and TP53) that were recurrent in at least two patients. By analysing the SCNAs, we found that the extent of chromosomal amplification was highly associated with the patient's cancer stage. Interestingly, 35% (6/17) of the patients had focal DNA amplifications in fibroblast growth factor receptor family genes. The integration of somatic SNVs, indels and SCNAs revealed significant alterations in the mitogen-activated protein kinase signalling pathways. CONCLUSIONS: Our genome-wide analysis of urachal cancer suggests that molecular characteristics may be important for the treatment of urachal cancer.

An R2* model of white matter for fiber orientation and myelin concentration.

Myelin, which consists of lipid bilayers, is one of the major constituents of white matter in the brain and has been suggested as a primary source of magnetic susceptibility contrasts. In this study, a new R2* model that simultaneously explains the effects of fiber orientation and myelin concentration is proposed and tested. In addition, a new approach that produces R2* maps without the effects of myelin is suggested. Experimental results demonstrate that the model reveals a high goodness of fit for the R2* distribution of white matter compared to a model that explains either fiber orientation or myelin concentration. The proposed R2* map shows a relatively uniform spatial distribution of R2* compared to the uncorrected R2* map and the fiber orientation or myelin concentration corrected R2* map.

Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system.

A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. The cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage.

Characterization of the deubiquitinating activity of USP19 and its role in endoplasmic reticulum-associated degradation.

Deubiquitinating enzymes (DUBs) regulate various cellular processes ranging from protein degradation to cellular signaling. USP19, the only DUB containing a carboxyl-terminal transmembrane domain, was proposed to function in endoplasmic reticulum-associated degradation (ERAD). Here we characterize the function and regulation of USP19. We identify Hsp90 as a specific partner that binds the catalytic domain of USP19 to promote substrate association. Intriguingly, although overexpressed USP19 interacts with Derlin-1 and other ERAD machinery factors in the membrane, endogenous USP19 is mostly in the cytosol where it binds Hsp90. Accordingly, we detect neither interaction of endogenous USP19 with Derlin-1 nor significant effect on ERAD by USP19 depletion. The USP19 transmembrane domain appears to be partially stabilized in the cytosol by an interaction with its own catalytic domain, resulting in auto-inhibition of its deubiquitinating activity. These results clarify the role of USP19 in ERAD and suggest a novel DUB regulation that involves chaperone association and membrane integration. Moreover, our study indicates that the localization of tail-anchored membrane proteins can be subject to regulation in cells.

RGS2 suppresses breast cancer cell growth via a MCPIP1-dependent pathway.

Regulator of G protein signaling 2 (RGS2) is a member of a family of proteins that functions as a GTPase-activating protein (GAP) for Gα subunits. RGS2 mRNA expression is lower in breast cancerous tissues than in normal tissues. In addition, expression of RGS2 is also lower in MCF7 (cancerous breast cells) than in MCF10A (normal breast cells). Here we investigated whether RGS2 inhibits growth of breast cancer cells. RGS2 overexpression in MCF7 cells inhibited epidermal growth factor- or serum-induced proliferation. In HEK293T cells expressing RGS2, cell growth was also significantly suppressed (In addition, exogenous expression of RGS2 in HEK293T cells resulted in the significant suppression of cell growth). These results suggest that RGS2 may have a tumor suppressor function. MG-132 treatment of MCF7 cells increased endogenous or exogenous RGS2 levels, suggesting a post-transcriptional regulatory mechanism that controls RGS2 protein levels. RGS2 protein was degraded polyubiquitinated the K71 residue, but stabilized by deubiquitinase monocyte chemotactic protein-induced protein 1 (MCPIP1), and not affected by dominant negative mutant (C157A) of MCPIP1. Gene expression profiling study showed that overexpression of RGS2 decreased levels of testis specific Y encoded like protein 5 (TSPYL5), which plays a causal role in breast oncogenesis. TSPYL5 protein expression was low in MCF10A and high in MCF7 cells, showing the opposite aspect to RGS2 expression. Additionally, RGS2 or MCPIP1 overexpression in MCF7 cells decreased TSPYL5 protein level, indicating that RGS2 stabilized by MCPIP1 have diminished TSPYL5 protein levels, thereby exerting an inhibitory effect of breast cancer cell growth.

Bag6/Bat3/Scythe: a novel chaperone activity with diverse regulatory functions in protein biogenesis and degradation.

Upon emerging from the ribosome exiting tunnel, polypeptide folding occurs immediately with the assistance of both ribosome-associated and free chaperones. While many chaperones known to date are dedicated folding catalysts, recent studies have revealed a novel chaperoning system that functions at the interface of protein biogenesis and quality control by using a special "holdase" activity in order to sort and channel client proteins to distinct destinations. The key component, Bag6/Bat3/Scythe, can effectively shield long hydrophobic segments exposed on the surface of a polypeptide, preventing aggregation or inappropriate interactions before a triaging decision is made. The biological consequences of Bag6-mediated chaperoning are divergent for different substrates, ranging from membrane integration to proteasome targeting and destruction. Accordingly, Bag6 can act in various cellular contexts in order to execute many essential cellular functions, while dysfunctions in the Bag6 system can cause severe cellular abnormalities that may be associated with some pathological conditions.

A ubiquitin-like domain recruits an oligomeric chaperone to a retrotranslocation complex in endoplasmic reticulum-associated degradation.

The Bag6-Ubl4A-Trc35 complex is a multifunctional chaperone that regulates various cellular processes. The diverse functions of Bag6 are supported by its ubiquitous localization to the cytoplasm, the nucleus, and membranes of the endoplasmic reticulum (ER) in cells. In ER-associated degradation (ERAD) pathways, Bag6 can interact with the membrane-associated ubiquitin ligase gp78 via its ubiquitin-like (UBL) domain, but the relative low affinity of this interaction does not reconcile with the fact that a fraction of Bag6 is tightly bound to the membranes. Here, we demonstrate that the UBL domain of Bag6 is required for interaction with the ER membranes. We find that in addition to gp78, the Bag6 UBL domain also binds a UBL-binding motif in UbxD8, an essential component of the gp78 ubiquitinating machinery. Importantly, Bag6 contains a proline-rich (PR) domain termed PDP (Proline rich-DUF3587-Proline rich) that forms homo-oligomer, allowing the UBL domain to form multivalent interactions with gp78 and UbxD8, which are essential for recruitment of Bag6 to the ER membrane. Furthermore, the PR domain comprises largely intrinsically disordered segments, which are sufficient for interaction with an unfolded substrate. We propose that simultaneous association with multiple ERAD factors helps to anchor a disordered chaperone oligomer to the site of retrotranslocation to prevent protein aggregation in ERAD.