MARCH5-dependent NLRP3 ubiquitination is required for mitochondrial NLRP3-NEK7 complex formation and NLRP3 inflammasome activation.

The NLRP3 inflammasome plays a key role in responding to pathogens, and endogenous damage and mitochondria are intensively involved in inflammasome activation. The NLRP3 inflammasome forms multiprotein complexes and its sequential assembly is important for its activation. Here, we show that NLRP3 is ubiquitinated by the mitochondria-associated E3 ligase, MARCH5. Myeloid cell-specific March5 conditional knockout (March5 cKO) mice failed to secrete IL-1ß and IL-18 and exhibited an attenuated mortality rate upon LPS or Pseudomonas aeruginosa challenge. Macrophages derived from March5 cKO mice also did not produce IL-1ß and IL-18 after microbial infection. Mechanistically, MARCH5 interacts with the NACHT domain of NLRP3 and promotes K27-linked polyubiquitination on K324 and K430 residues of NLRP3. Ubiquitination-defective NLRP3 mutants on K324 and K430 residues are not able to bind to NEK7, nor form NLRP3 oligomers leading to abortive ASC speck formation and diminished IL-1ß production. Thus, MARCH5-dependent NLRP3 ubiquitination on the mitochondria is required for NLRP3-NEK7 complex formation and NLRP3 oligomerization. We propose that the E3 ligase MARCH5 is a regulator of NLRP3 inflammasome activation on the mitochondria.

GPDminer: a tool for extracting named entities and analyzing relations in biological literature.

PURPOSE: The expansion of research across various disciplines has led to a substantial increase in published papers and journals, highlighting the necessity for reliable text mining platforms for database construction and knowledge acquisition. This abstract introduces GPDMiner(Gene, Protein, and Disease Miner), a platform designed for the biomedical domain, addressing the challenges posed by the growing volume of academic papers. METHODS: GPDMiner is a text mining platform that utilizes advanced information retrieval techniques. It operates by searching PubMed for specific queries, extracting and analyzing information relevant to the biomedical field. This system is designed to discern and illustrate relationships between biomedical entities obtained from automated information extraction. RESULTS: The implementation of GPDMiner demonstrates its efficacy in navigating the extensive corpus of biomedical literature. It efficiently retrieves, extracts, and analyzes information, highlighting significant connections between genes, proteins, and diseases. The platform also allows users to save their analytical outcomes in various formats, including Excel and images. CONCLUSION: GPDMiner offers a notable additional functionality among the array of text mining tools available for the biomedical field. This tool presents an effective solution for researchers to navigate and extract relevant information from the vast unstructured texts found in biomedical literature, thereby providing distinctive capabilities that set it apart from existing methodologies. Its application is expected to greatly benefit researchers in this domain, enhancing their capacity for knowledge discovery and data management.

Regulation of nuclear DNA damage response by mitochondrial morphofunctional pathway.

Cells are constantly challenged by genotoxic stresses that can lead to genome instability. The integrity of the nuclear genome is preserved by the DNA damage response (DDR) and repair. Additionally, these stresses can induce mitochondria to transiently hyperfuse; however, it remains unclear whether canonical DDR is linked to these mitochondrial morphological changes. Here, we report that the abolition of mitochondrial fusion causes a substantial defect in the ATM-mediated DDR signaling. This deficiency is overcome by the restoration of mitochondria fusion. In cells with fragmented mitochondria, genotoxic stress-induced activation of JNK and its translocation to DNA lesion are lost. Importantly, the mitochondrial fusion machinery of MFN1/MFN2 associates with Sab (SH3BP5) and JNK, and these interactions are indispensable for the Sab-mediated activation of JNK and the ATM-mediated DDR signaling. Accordingly, the formation of BRCA1 and 53BP1 foci, as well as homology and end-joining repair are impaired in cells with fragmented mitochondria. Together, these data show that mitochondrial fusion-dependent JNK signaling is essential for the DDR, providing vital insight into the integration of nuclear and cytoplasmic stress signals.

CREB mediates the C. elegans dauer polyphenism through direct and cell-autonomous regulation of TGF-ß expression.

Animals can adapt to dynamic environmental conditions by modulating their developmental programs. Understanding the genetic architecture and molecular mechanisms underlying developmental plasticity in response to changing environments is an important and emerging area of research. Here, we show a novel role of cAMP response element binding protein (CREB)-encoding crh-1 gene in developmental polyphenism of C. elegans. Under conditions that promote normal development in wild-type animals, crh-1 mutants inappropriately form transient pre-dauer (L2d) larvae and express the L2d marker gene. L2d formation in crh-1 mutants is specifically induced by the ascaroside pheromone ascr#5 (asc-ωC3; C3), and crh-1 functions autonomously in the ascr#5-sensing ASI neurons to inhibit L2d formation. Moreover, we find that CRH-1 directly binds upstream of the daf-7 TGF-ß locus and promotes its expression in the ASI neurons. Taken together, these results provide new insight into how animals alter their developmental programs in response to environmental changes.

De novo phosphorylation of H2AX by WSTF regulates transcription-coupled homologous recombination repair.

Histone H2AX undergoes a phosphorylation switch from pTyr142 (H2AX-pY142) to pSer139 (γH2AX) in the DNA damage response (DDR); however, the functional role of H2AX-pY142 remains elusive. Here, we report a new layer of regulation involving transcription-coupled H2AX-pY142 in the DDR. We found that constitutive H2AX-pY142 generated by Williams-Beuren syndrome transcription factor (WSTF) interacts with RNA polymerase II (RNAPII) and is associated with RNAPII-mediated active transcription in proliferating cells. Also, removal of pre-existing H2AX-pY142 by ATM-dependent EYA1/3 phosphatases disrupts this association and requires for transcriptional silencing at transcribed active damage sites. The following recovery of H2AX-pY142 via translocation of WSTF to DNA lesions facilitates transcription-coupled homologous recombination (TC-HR) in the G1 phase, whereby RAD51 loading, but not RPA32, utilizes RNAPII-dependent active RNA transcripts as donor templates. We propose that the WSTF-H2AX-RNAPII axis regulates transcription and TC-HR repair to maintain genome integrity.

Glucocorticoids alleviate particulate matter-induced COX-2 expression and mitochondrial dysfunction through the Bcl-2/GR complex in A549 cells.

Exposure to particulate matter (PM) causes mitochondrial dysfunction and lung inflammation. The cyclooxygenase-2 (COX-2) pathway is important for inflammation and mitochondrial function. However, the mechanisms by which glucocorticoid receptors (GRs) suppress COX-2 expression during PM exposure have not been elucidated yet. Hence, we examined the mechanisms underlying the dexamethasone-mediated suppression of the PM-induced COX-2/prostaglandin E2 (PGE2) pathway in A549 cells. The PM-induced increase in COX-2 protein, mRNA, and promoter activity was suppressed by glucocorticoids; this effect of glucocorticoids was antagonized by the GR antagonist RU486. COX-2 induction was correlated with the ability of PM to increase reactive oxygen species (ROS) levels. Consistent with this, antioxidant treatment significantly abolished COX-2 induction, suggesting that ROS is involved in PM-mediated COX-2 induction. We also observed a low mitochondrial membrane potential in PM-treated A549 cells, which was reversed by dexamethasone. Moreover, glucocorticoids significantly enhanced Bcl-2/GR complex formation in PM-treated A549 cells. Glucocorticoids regulate the PM-exposed induction of COX-2 expression and mitochondrial dysfunction and increase the interaction between GR and Bcl-2. These findings suggest that the COX-2/PGE2 pathway and the interaction between GR and Bcl-2 are potential key therapeutic targets for the suppression of inflammation under PM exposure.

Mitochondrial E3 ligase MARCH5 is a safeguard against DNA-PKcs-mediated immune signaling in mitochondria-damaged cells.

Mitochondrial dysfunction is important in various chronic degenerative disorders, and aberrant immune responses elicited by cytoplasmic mitochondrial DNA (mtDNA) may be related. Here, we developed mtDNA-targeted MTERF1-FokI and TFAM-FokI endonuclease systems to induce mitochondrial DNA double-strand breaks (mtDSBs). In these cells, the mtDNA copy number was significantly reduced upon mtDSB induction. Interestingly, in cGAS knockout cells, synthesis of interferon ß1 and interferon-stimulated gene was increased upon mtDSB induction. We found that mtDSBs activated DNA-PKcs and HSPA8 in a VDAC1-dependent manner. Importantly, the mitochondrial E3 ligase MARCH5 bound active DNA-PKcs in cells with mtDSBs and reduced the type І interferon response through the degradation of DNA-PKcs. Likewise, mitochondrial damage caused by LPS treatment in RAW264.7 macrophage cells increased phospho-HSPA8 levels and the synthesis of mIFNB1 mRNA in a DNA-PKcs-dependent manner. Accordingly, in March5 knockout macrophages, phospho-HSPA8 levels and the synthesis of mIFNB1 mRNA were prolonged after LPS stimulation. Together, cytoplasmic mtDNA elicits a cellular immune response through DNA-PKcs, and mitochondrial MARCH5 may be a safeguard to prevent persistent inflammatory reactions.

A single chemosensory GPCR is required for a concentration-dependent behavioral switching in C. elegans.

Animals detect and discriminate countless environmental chemicals for their well-being and survival. Although a single chemical can trigger opposing behavioral responses depending on its concentration, the mechanisms underlying such a concentration-dependent switching remain poorly understood. Here, we show that C. elegans exhibits either attraction or avoidance of the bacteria-derived volatile chemical dimethyl trisulfide (DMTS) depending on its concentration. This behavioral switching is mediated by two different types of chemosensory neurons, both of which express the DMTS-sensitive seven-transmembrane G protein-coupled receptor (GPCR) SRI-14. These two sensory neurons share downstream interneurons that process and translate DMTS signals via distinct glutamate receptors to generate the appropriate behavioral outcome. Thus, our results present one mechanism by which an animal connects two distinct types of chemosensory neurons detecting a common ligand to alternate downstream circuitry, thus efficiently switching between specific behavioral programs based on ligand concentration.

Leisure Sports Participants' Engagement in Preventive Health Behaviors and Their Experience of Constraints on Performing Leisure Activities During the COVID-19 Pandemic.

This study assessed the demographic characteristics of Koreans engaged in leisure sports activities during the COVID-19 pandemic and the differences in their preventive health behaviors and constraints on leisure activities. For this study, the demographic characteristics (gender, age, marital status, level of participation in leisure sports, years of participation, companions with whom individuals participating in these sports, type of space used for performing the sports, occupation, and average monthly income) of 544 leisure sport participants (men: 46.0%, women: 54.0%; average age: 36.8 and 33.5 years, respectively), who were recruited on a nationwide basis, were examined through an online survey. Then, comparisons between groups were performed using independent t-tests, one-way analysis of variance, and multivariate analysis of variance. Women who participated in both indoor and outdoor leisure sports showed higher adoption of health prevention behaviors than their male counterparts, and married individuals who participated in indoor leisure sports showed higher adoption of health prevention behaviors than unmarried participants. Moreover, individuals who participated in both indoor and outdoor leisure sports by themselves had many interpersonal constraints overall, and the group of married individuals who participated in indoor leisure sports showed structural constraints. In conclusion, leisure sports participants have adopted many health prevention behaviors during the COVID-19 pandemic, but this had led to some interpersonal constraints. These results indicate that, in the case of future pandemics, personal and institutional efforts will need to be made to promote participation in leisure sports and prevent excessive social isolation.

Dual targeting of RIG-I and MAVS by MARCH5 mitochondria ubiquitin ligase in innate immunity.

The mitochondrial antiviral signaling (MAVS) protein on the mitochondrial outer membrane acts as a central signaling molecule in the RIG-I-like receptor (RLR) signaling pathway by linking upstream viral RNA recognition to downstream signal activation. We previously reported that mitochondrial E3 ubiquitin ligase, MARCH5, degrades the MAVS protein aggregate and prevents persistent downstream signaling. Since the activated RIG-I oligomer interacts and nucleates the MAVS aggregate, MARCH5 might also target this oligomer. Here, we report that MARCH5 targets and degrades RIG-I, but not its inactive phosphomimetic form (RIG-IS8E). The MARCH5-mediated reduction of RIG-I is restored in the presence of MG132, a proteasome inhibitor. Upon poly(I:C) stimulation, RIG-I forms an oligomer and co-expression of MARCH5 reduces the expression of this oligomer. The RING domain of MARCH5 is necessary for binding to the CARD domain of RIG-I. In an in vivo ubiquitination assay, MARCH5 transfers the Lys 48-linked polyubiquitin to Lys 193 and 203 residues of RIG-I. Thus, dual targeting of active RIG-I and MAVS protein oligomers by MARCH5 is an efficient way to switch-off RLR signaling. We propose that modulation of MARCH5 activity might be beneficial for the treatment of chronic immune diseases.

Mitochondria ubiquitin ligase, MARCH5 resolves hepatitis B virus X protein aggregates in the liver pathogenesis.

Infection of hepatitis B virus (HBV) increase the incidence of chronic liver disease and hepatocellular carcinoma (HCC). The hepatitis B viral x (HBx) protein encoded by the HBV genome contributes to the pathogenesis of HCC and thus, negative regulation of HBx is beneficial for the alleviation of the disease pathogenesis. MARCH5 is a mitochondrial E3 ubiquitin ligase and here, we show that high MARCH5 expression levels are correlated with improved survival in HCC patients. MARCH5 interacts with HBx protein mainly accumulated in mitochondria and targets it for degradation. The N-terminal RING domain of MARCH5 was required for the interaction with HBx, and MARCH5H43W lacking E3 ligase activity failed to reduce HBx protein levels. High expression of HBx results in the formation of protein aggregates in semi-denaturing detergent agarose gels and MARCH5 mediates the elimination of protein aggregates through the proteasome pathway. HBx-induced ROS production, mitophagy, and cyclooxygenase-2 gene expression were suppressed in the presence of high MARCH5 expression. These results suggest MARCH5 as a target for alleviating HBV-mediated liver disease.

DOT1L cooperates with the c-Myc-p300 complex to epigenetically derepress CDH1 transcription factors in breast cancer progression.

DOT1L has emerged as an anticancer target for MLL-associated leukaemias; however, its functional role in solid tumours is largely unknown. Here we identify that DOT1L cooperates with c-Myc and p300 acetyltransferase to epigenetically activate epithelial-mesenchymal transition (EMT) regulators in breast cancer progression. DOT1L recognizes SNAIL, ZEB1 and ZEB2 promoters via interacting with the c-Myc-p300 complex and facilitates lysine-79 methylation and acetylation towards histone H3, leading to the dissociation of HDAC1 and DNMT1 in the regions. The upregulation of these EMT regulators by the DOT1L-c-Myc-p300 complex enhances EMT-induced breast cancer stem cell (CSC)-like properties. Furthermore, in vivo orthotopic xenograft models show that DOT1L is required for malignant transformation of breast epithelial cells and breast tumour initiation and metastasis. Clinically, DOT1L expression is associated with poorer survival and aggressiveness of breast cancers. Collectively, we suggest that cooperative effect of DOT1L and c-Myc-p300 is critical for acquisition of aggressive phenotype of breast cancer by promoting EMT/CSC.