HERC3 facilitates ERAD of select membrane proteins by recognizing membrane-spanning domains.

Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 uniquely promotes CFTR ERAD. In vitro assay revealed that HERC3 directly interacts with the exposed membrane-spanning domains (MSDs) of CFTR but not with the MSDs embedded in liposomes. Therefore, HERC3 could play a role in the quality control of MSDs in the cytoplasm and might be crucial for the ERAD pathway of select membrane proteins.

Membrane-Associated Ubiquitin Ligase RING Finger Protein 152 Orchestrates Melanogenesis via Tyrosinase Ubiquitination.

Lysosomal degradation of tyrosinase, a pivotal enzyme in melanin synthesis, negatively impacts melanogenesis in melanocytes. Nevertheless, the precise molecular mechanisms by which lysosomes target tyrosinase have remained elusive. Here, we identify RING (Really Interesting New Gene) finger protein 152 (RNF152) as a membrane-associated ubiquitin ligase specifically targeting tyrosinase for the first time, utilizing AlphaScreen technology. We observed that modulating RNF152 levels in B16 cells, either via overexpression or siRNA knockdown, resulted in decreased or increased levels of both tyrosinase and melanin, respectively. Notably, RNF152 and tyrosinase co-localized at the trans-Golgi network (TGN). However, upon treatment with lysosomal inhibitors, both proteins appeared in the lysosomes, indicating that tyrosinase undergoes RNF152-mediated lysosomal degradation. Through ubiquitination assays, we found the indispensable roles of both the RING and transmembrane (TM) domains of RNF152 in facilitating tyrosinase ubiquitination. In summary, our findings underscore RNF152 as a tyrosinase-specific ubiquitin ligase essential for regulating melanogenesis in melanocytes.

SRPKIN-1 as an inhibitor against hepatitis B virus blocking the viral particle formation and the early step of the viral infection.

New antiviral agents are needed for the treatment of hepatitis B virus (HBV) infection because currently available drugs do not completely eradicate chronic HBV in patients. Phosphorylation dynamics of the HBV core protein (HBc) regulate several processes in the HBV life cycle, including nucleocapsid formation, cell trafficking, and virus uncoating after entry. In this study, the SRPK inhibitors SPHINX31, SRPIN340, and SRPKIN-1 showed concentration-dependent anti-HBV activity. Detailed analysis of the effects of SRPKIN-1, which exhibited the strongest inhibitory activity, on the HBV replication process showed that it inhibits the formation of infectious particles by inhibiting pregenomic RNA packaging into capsids and nucleocapsid envelopment. Mass spectrometry analysis combined with cell-free translation system experiments revealed that hyperphosphorylation of the C-terminal domain of HBc is inhibited by SRPKIN-1. Further, SRPKIN-1 exhibited concentration-dependent inhibition of HBV infection not only in HepG2-hNTCP-C4 cells but also in fresh human hepatocytes (PXB cells) and in the single-round infection system. Treatment with SRPKIN-1 at the time of infection reduced the nuclease sensitivity of HBV DNA in the nuclear fraction. These results suggest that SRPKIN-1 has the potential to not only inhibit the HBV particle formation process but also impair the early stages of viral infection.

A strategy for orthogonal deubiquitination using a bump-and-hole approach.

We have successfully applied a bump-and-hole approach to establish orthogonal deubiquitination in which a ubiquitin substrate variant is specifically targeted by an engineered deubiquitinating enzyme (DUB). This makes it possibe to selectively observe and measure a single type of DUB activity in living cells.

Wnt activation disturbs cell competition and causes diffuse invasion of transformed cells through NF-κB-MMP21 pathway.

Normal epithelial cells exert their competitive advantage over RasV12-transformed cells and eliminate them into the apical lumen via cell competition. However, the internal or external factors that compromise cell competition and provoke carcinogenesis remain elusive. In this study, we examine the effect of sequential accumulation of gene mutations, mimicking multi-sequential carcinogenesis on RasV12-induced cell competition in intestinal epithelial tissues. Consequently, we find that the directionality of RasV12-cell extrusion in Wnt-activated epithelia is reversed, and transformed cells are delaminated into the basal lamina via non-cell autonomous MMP21 upregulation. Subsequently, diffusively infiltrating, transformed cells develop into highly invasive carcinomas. The elevated production of MMP21 is elicited partly through NF-κB signaling, blockage of which restores apical elimination of RasV12 cells. We further demonstrate that the NF-κB-MMP21 axis is significantly bolstered in early colorectal carcinoma in humans. Collectively, this study shows that cells with high mutational burdens exploit cell competition for their benefit by behaving as unfit cells, endowing them with an invasion advantage.

Role of hepcidin upregulation and proteolytic cleavage of ferroportin 1 in hepatitis C virus-induced iron accumulation.

Hepatitis C virus (HCV) is a pathogen characterized not only by its persistent infection leading to the development of cirrhosis and hepatocellular carcinoma (HCC), but also by metabolic disorders such as lipid and iron dysregulation. Elevated iron load is commonly observed in the livers of patients with chronic hepatitis C, and hepatic iron overload is a highly profibrogenic and carcinogenic factor that increases the risk of HCC. However, the underlying mechanisms of elevated iron accumulation in HCV-infected livers remain to be fully elucidated. Here, we observed iron accumulation in cells and liver tissues under HCV infection and in mice expressing viral proteins from recombinant adenoviruses. We established two molecular mechanisms that contribute to increased iron load in cells caused by HCV infection. One is the transcriptional induction of hepcidin, the key hormone for modulating iron homeostasis. The transcription factor cAMP-responsive element-binding protein hepatocyte specific (CREBH), which was activated by HCV infection, not only directly recognizes the hepcidin promoter but also induces bone morphogenetic protein 6 (BMP6) expression, resulting in an activated BMP-SMAD pathway that enhances hepcidin promoter activity. The other is post-translational regulation of the iron-exporting membrane protein ferroportin 1 (FPN1), which is cleaved between residues Cys284 and Ala285 in the intracytoplasmic loop region of the central portion mediated by HCV NS3-4A serine protease. We propose that host transcriptional activation triggered by endoplasmic reticulum stress and FPN1 cleavage by viral protease work in concert to impair iron efflux, leading to iron accumulation in HCV-infected cells.

The E3 ligase DTX2 inhibits RUNX1 function by binding its C terminus and prevents the growth of RUNX1-dependent leukemia cells.

Transcription factor RUNX1 plays important roles in hematopoiesis and leukemogenesis. RUNX1 function is tightly controlled through posttranslational modifications, including ubiquitination and acetylation. However, its regulation via ubiquitination, especially proteasome-independent ubiquitination, is poorly understood. We previously identified DTX2 as a RUNX1-interacting E3 ligase using a cell-free AlphaScreen assay. In this study, we examined whether DTX2 is involved in the regulation of RUNX1 using in vitro and ex vivo analyses. DTX2 bound to RUNX1 and other RUNX family members RUNX2 and RUNX3 through their C-terminal region. DTX2-induced RUNX1 ubiquitination did not result in RUNX1 protein degradation. Instead, we found that the acetylation of RUNX1, which is known to enhance the transcriptional activity of RUNX1, was inhibited in the presence of DTX2. Concomitantly, DTX2 reduced the RUNX1-induced activation of an MCSFR luciferase reporter. We also found that DTX2 induced RUNX1 cytoplasmic mislocalization. Moreover, DTX2 overexpression showed a substantial growth-inhibitory effect in RUNX1-dependent leukemia cell lines. Thus, our findings indicate a novel aspect of the ubiquitination and acetylation of RUNX1 that is modulated by DTX2 in a proteosome-independent manner.

A girl with membranous nephropathy associated with ventriculoperitoneal shunt infection.

Glomerulopathy associated with shunt infection is commonly membranoproliferative glomerulonephritis, whereas the causative organisms of secondary membranous nephropathy are usually viruses. We report a case of membranous nephropathy associated with shunt infection. The patient was born at 29-week gestation with a birth weight of 1178 g. Ventriculoperitoneal shunt surgery had been performed for congenital hydrocephalus. Thereafter, she had experienced seven shunt infections. At the age 13 years, proteinuria was detected in a school urinary screening. Urinalysis at our hospital demonstrated 3 + protein and 3 + blood. Laboratory testing demonstrated a serum creatinine 0.5 m/dl, albumin 2.5 g/dl, C-reactive protein (CRP) 13.7 mg/dl, and C3 182 mg/dl. Prior to repeat urinalysis, the patient developed vomiting and was admitted with suspected shunt infection. On admission, her body temperature was 36.0 ºC. Physical examination was unremarkable other than small stature and a palpable mass in the left upper quadrant. Urinalysis demonstrated 2 + protein and 1 + blood with no cells or casts. The urinary protein excretion was 3 g/day. Abnormal laboratory tests included erythrocyte sedimentation rate 102 mm/hr, CRP 11.67 mg/dl, IgG 2442 mg/dl, C3 177 mg/dl, and C4 44 mg/dl. Antibiotic therapy was initiated for a presumptive diagnosis of shunt infection and the shunt catheter was removed. Cultures obtained after antibiotic administration were negative. Proteinuria persisted after control of the shunt infection. Histology of a renal biopsy demonstrated membranous nephropathy with diffuse granular IgG staining and subepithelial deposits. Three possible pathomechanisms for her membranous nephropathy were considered.

OTUD1 deubiquitinase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways.

Deubiquitinating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin chain assembly complex (LUBAC)-induced NF-κB activation, and identified OTUD1 as a potent suppressor. OTUD1 regulates the canonical NF-κB pathway by hydrolyzing K63-linked ubiquitin chains from NF-κB signaling factors, including LUBAC. OTUD1 negatively regulates the canonical NF-κB activation, apoptosis, and necroptosis, whereas OTUD1 upregulates the interferon (IFN) antiviral pathway. Mass spectrometric analysis showed that OTUD1 binds KEAP1, and the N-terminal intrinsically disordered region of OTUD1, which contains an ETGE motif, is indispensable for the KEAP1-binding. Indeed, OTUD1 is involved in the KEAP1-mediated antioxidant response and reactive oxygen species (ROS)-induced cell death, oxeiptosis. In Otud1-/--mice, inflammation, oxidative damage, and cell death were enhanced in inflammatory bowel disease, acute hepatitis, and sepsis models. Thus, OTUD1 is a crucial regulator for the inflammatory, innate immune, and oxidative stress responses and ROS-associated cell death pathways.

Unsupervised learning architecture for classifying the transient noise of interferometric gravitational-wave detectors.

In the data obtained by laser interferometric gravitational wave detectors, transient noise with non-stationary and non-Gaussian features occurs at a high rate. This often results in problems such as detector instability and the hiding and/or imitation of gravitational-wave signals. This transient noise has various characteristics in the time-frequency representation, which is considered to be associated with environmental and instrumental origins. Classification of transient noise can offer clues for exploring its origin and improving the performance of the detector. One approach for accomplishing this is supervised learning. However, in general, supervised learning requires annotation of the training data, and there are issues with ensuring objectivity in the classification and its corresponding new classes. By contrast, unsupervised learning can reduce the annotation work for the training data and ensure objectivity in the classification and its corresponding new classes. In this study, we propose an unsupervised learning architecture for the classification of transient noise that combines a variational autoencoder and invariant information clustering. To evaluate the effectiveness of the proposed architecture, we used the dataset (time-frequency two-dimensional spectrogram images and labels) of the Laser Interferometer Gravitational-wave Observatory (LIGO) first observation run prepared by the Gravity Spy project. The classes provided by our proposed unsupervised learning architecture were consistent with the labels annotated by the Gravity Spy project, which manifests the potential for the existence of unrevealed classes.

Polymorphism and optical, magnetic and thermal properties of the either phyllo- or inosilicate-analogous borosulfate Cu[B2(SO4)4].

Two polymorphs of the borosulfate Cu[B2(SO4)4] can be selectively prepared by solvothermal syntheses. The crystal structures of inosilicate-analogous α-Cu[B2(SO4)4] (P1̄, no. 2, a = 5.2636(2), b = 7.1449(2), c = 7.9352(2) Å, α = 73.698(2)°, ß = 70.737(2)°, γ = 86.677(2)°, 65 parameters, RBragg = 0.0052) and the new phyllosilicate-analogous polymorph ß-Cu[B2(SO4)4] (P21/n, no. 14, a = 7.712(3), b = 8.149(3), c = 9.092(3) Å, ß = 111.22(1)°, 3829 independent reflections, 106 parameters, wR2 = 0.054) are discussed. Further, the optical, magnetic and thermal properties of both polymorphs are investigated with focus on the role of the Cu2+ cation and its Jahn-Teller effect. The findings are confirmed by DFT calculations yielding insights in the stability of the synthesised polymorphs as well as a predicted γ-modification. Additionally, the crystal structures of two polymorphs of copper hydrogensulfate Cu(HSO4)2-I (P21/n, no. 14, a = 4.7530(2), b = 8.5325(4), c = 7.3719(3) Å, ß = 100.063(1)°, 1063 independent reflections, 55 parameters, wR2 = 0.052) and Cu(HSO4)2-II (P1̄, no. 2, a = 4.79.88(8), b = 7.857(1), c = 8.057(1) Å, α = 77.86(1)°, ß = 87.02(1)°, γ = 89.82(1)°, 1044 independent reflections, 109 parameters, wR2 = 0.132) as well as that of Cu[S2O7] (C2/c, no. 15, a = 6.6341(4), b = 8.7302(5), c = 9.0555(8) Å, ß = 104.763(3)°, 1117 independent reflections, 48 parameters, wR2 = 0.049) are presented and the cyclosilicate-analogous borosulfate Cu[B(SO4)2(HSO4)] is fully characterised with respect to its optical and thermal properties.

A simple method for labeling proteins and antibodies with biotin using the proximity biotinylation enzyme TurboID.

Proteins and antibodies labeled with biotin have been widely used for protein analysis, enzyme immunoassays, and diagnoses. Presently, they are prepared using either a chemical reaction involving a biotin N-hydroxysuccinimide (NHS) ester compound or by enzymatic biotin ligation using a combination of a biotinylation-peptide tag and Escherichia coli BirA. However, these methods are relatively complicated. Recently BirA was improved to TurboID, a highly active enzyme for proximity labeling with biotin. Here, we demonstrate a novel simple biotin labeling method for proteins and antibodies using TurboID. Purified TurboID was mixed with a protein or an antibody in the presence of biotin and ATP in the general biochemical buffer condition, followed by biotin labeling. Biotin labeling sites by TurboID were found on the surface of green fluorescent protein. Biotin labeling of IκBα by TurboID indicated its binding to RelA. Furthermore, TurboID-dependent biotin labeling of monoclonal antibodies from rabbits and mice could be directly used for immunoblotting detection of specific proteins without the purification step. These results indicate that TurboID provides a very useful and simple method for biotin labeling of functional proteins.

Inattentive Driving Detection Using Body-Worn Sensors: Feasibility Study.

This study aims to build a system for detecting a driver's internal state using body-worn sensors. Our system is intended to detect inattentive driving that occurs during long-term driving on a monotonous road, such as a high-way road. The inattentive state of a driver in this study is an absent-minded state caused by a decrease in driver vigilance levels due to fatigue or drowsiness. However, it is difficult to clearly define these inattentive states because it is difficult for the driver to recognize when they fall into an absent-minded state. To address this problem and achieve our goal, we have proposed a detection algorithm for inattentive driving that not only uses a heart rate sensor, but also uses body-worn inertial sensors, which have the potential to detect driver behavior more accurately and at a much lower cost. The proposed method combines three detection models: body movement, drowsiness, and inattention detection, based on an anomaly detection algorithm. Furthermore, we have verified the accuracy of the algorithm with the experimental data for five participants that were measured in long-term and monotonous driving scenarios by using a driving simulator. The results indicate that our approach can detect both the inattentive and drowsiness states of drivers using signals from both the heart rate sensor and accelerometers placed on wrists.

SIRVVD model-based verification of the effect of first and second doses of COVID-19/SARS-CoV-2 vaccination in Japan.

As of August 2021, COVID-19 is still spreading in Japan. Vaccination, one of the key measures to bring COVID-19 under control, began in February 2021. Previous studies have reported that COVID-19 vaccination reduces the number of infections and mortality rates. However, simulations of spreading infection have suggested that vaccination in Japan is insufficient. Therefore, we developed a susceptible-infected-recovered-vaccination1-vaccination2-death model to verify the effect of the first and second vaccination doses on reducing the number of infected individuals in Japan; this includes an infection simulation. The results confirm that appropriate vaccination measures will sufficiently reduce the number of infected individuals and reduce the mortality rate.

Protein-protein interactions between jasmonate-related master regulator MYC and transcriptional mediator MED25 depend on a short binding domain.

A network of protein-protein interactions (PPI) is involved in the activation of (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), a plant hormone that regulates plant defense responses as well as plant growth and development. In the absence of JA-Ile, inhibitory protein jasmonate-ZIM-domain (JAZ) represses JA-related transcription factors, including a master regulator, MYC. In contrast, when JA-Ile accumulates in response to environmental stresses, PPI occurs between JAZ and the F-box protein COI1, which triggers JAZ degradation, resulting in derepressed MYC that can interact with the transcriptional mediator MED25 and upregulate JA-Ile-related gene expression. Activated JA signaling is eventually suppressed through the catabolism of JA-Ile and feedback suppression by JAZ splice variants containing a cryptic MYC-interacting domain (CMID). However, the detailed structural basis of some PPIs involved in JA-Ile signaling remains unclear. Herein, we analyzed PPI between MYC3 and MED25, focusing on the key interactions that activate the JA-Ile signaling pathway. Biochemical assays revealed that a short binding domain of MED25 (CMIDM) is responsible for the interaction with MYC, and that a bipartite interaction is critical for the formation of a stable complex. We also show the mode of interaction between MED25 and MYC is closely related to that of CMID and MYC. In addition, quantitative analyses on the binding of MYC3-JAZs and MYC3-MED25 revealed the order of binding affinity as JAZJas < MED25CMIDM < JAZCMID, suggesting a mechanism for how the transcriptional machinery causes activation and negative feedback regulation during jasmonate signaling. These results further illuminate the transcriptional machinery responsible for JA-Ile signaling.

Fast screening framework for infection control scenario identification.

Due to the emergence of the novel coronavirus disease, many recent studies have investigated prediction methods for infectious disease transmission. This paper proposes a framework to quickly screen infection control scenarios and identify the most effective scheme for reducing the number of infected individuals. Analytical methods, as typified by the SIR model, can conduct trial-and-error verification with low computational costs; however, they must be reformulated to introduce additional constraints, and thus are inappropriate for case studies considering detailed constraint parameters. In contrast, multi-agent system (MAS) simulators introduce detailed parameters but incur high computation costs per simulation, making them unsuitable for extracting effective measures. Therefore, we propose a framework that implements an MAS for constructing a training dataset, and then trains a support vector regression (SVR) model to obtain effective measure results. The proposed framework overcomes the weaknesses of conventional methods to produce effective control measure recommendations. The constructed SVR model was experimentally verified by comparing its performance on datasets with expected and unexpected outputs. Although datasets producing an unexpected output decreased the prediction accuracy, by removing randomness from the training dataset, the accuracy of the proposed method was still high in these cases. High-precision predictions of the MAS-based simulation output were obtained for both test datasets in under one second of the computational time. Furthermore, the experimental results establish that the proposed framework can obtain intuitively correct outputs for unknown inputs, and produces sufficiently high-precision prediction with lower computation costs than an existing method.

SIR model-based verification of effect of COVID-19 Contact-Confirming Application (COCOA) on reducing infectors in Japan.

As of April 2021, the coronavirus disease (COVID-19) continues to spread in Japan. To overcome COVID-19, the Ministry of Health, Labor, and Welfare of the Japanese government developed and released the COVID-19 Contact-Confirming Application (COCOA) on June 19, 2020. COCOA users can know whether they have come into contact with infectors. If persons who receive a contact notification through COCOA undertake self-quarantine, the number of infectors in Japan will decrease. However, the effectiveness of COCOA in reducing the number of infectors depends on the usage rate of COCOA, the rate of fulfillment of contact condition, the rate of undergoing the reverse transcription polymerase chain reaction (RT-PCR) test, the false negative rate of the RT-PCR test, the rate of infection registration, and the self-quarantine rate. Therefore, we developed a Susceptible-Infected-Removed (SIR) model to estimate the effectiveness of COCOA. In this paper, we introduce the SIR model and report the simulation results for different scenarios that were assumed for Japan.

Myofiber androgen receptor increases muscle strength mediated by a skeletal muscle splicing variant of Mylk4.

Androgens have a robust effect on skeletal muscles to increase muscle mass and strength. The molecular mechanism of androgen/androgen receptor (AR) action on muscle strength is still not well known, especially for the regulation of sarcomeric genes. In this study, we generated androgen-induced hypertrophic model mice, myofiber-specific androgen receptor knockout (cARKO) mice supplemented with dihydrotestosterone (DHT). DHT treatment increased grip strength in control mice but not in cARKO mice. Transcriptome analysis by RNA-seq, using skeletal muscles obtained from control and cARKO mice treated with or without DHT, identified a fast-type muscle-specific novel splicing variant of Myosin light-chain kinase 4 (Mylk4) as a target of AR in skeletal muscles. Mylk4 knockout mice exhibited decreased maximum isometric torque of plantar flexion and passive stiffness of myofibers due to reduced phosphorylation of Myomesin 1 protein. This study suggests that androgen-induced skeletal muscle strength is mediated with Mylk4 and Myomesin 1 axis.

Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neosubstrate PLZF.

Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)-containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide- and 5-hydroxythalidomide-induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell-free protein synthesis system, PLZF was identified as a thalidomide-dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4CRBN in complex with thalidomide, its derivatives or 5-hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5-hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide-dependent CRBN substrate SALL4, was induced by thalidomide or 5-hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide-induced phenotypes. Our findings implicate PLZF as an important thalidomide-induced CRBN neosubstrate involved in thalidomide teratogenicity.