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Abstract : Inositol phosphates are water-soluble intracellular signaling molecules found in eukaryotes from yeasts to mammals, which are synthesized by a complex network of enzymes including inositol phosphate kinases. Among these, inositol polyphosphate multikinase (IPMK) is a promiscuous enzyme with broad substrate specificity, which phosphorylates multiple inositol phosphates, as well as phosphatidylinositol 4,5-bisphosphate. In addition to its catalytic actions, IPMK is known to non-catalytically control major signaling events via direct protein-protein interactions. In this review, we describe the general characteristics of IPMK, highlight its pleiotropic roles in various physiological and pathological conditions, and discuss future challenges in the field of IPMK signaling pathways.

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Abstract : Brain disease is known to cause irrevocable and fatal loss of biological function once damaged. One of various causes of its development is damage to neuron cells caused by hyperactivated microglia, which function as immune cells in brain. Among the genes expressed in microglia stimulated by various antigens, annexin A1 (ANXA1) is expressed in the early phase of the inflammatory response and plays an important role in controlling the immune response. In this study, we assessed whether ANXA1 can be a therapeutic target gene for the initial reduction of the immune response induced by microglia to minimize neuronal damage. To address this, mouse-origin microglial cells were stimulated to mimic an immune response by lipopolysaccharide (LPS) treatment. The LPS treatment caused activation of ANXA1 gene and expression of inflammatory cytokines. To assess the biological function in microglia by the downregulation of ANXA1 gene, cells were treated with short hairpin RNA-ANXA1. Downregulated ANXA1 affected the function of mitochondria in the microglia and showed reduced neuronal damage when compared to the control group in the co-culture system. Taken together, our results showed that ANXA1 could be used as a potential therapeutic target for inflammation-related neurodegenerative diseases.

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Abstract : In eukaryotic cells, organelles are distributed and positioned in proximity to each other through molecular tether proteins. Among these, the mitochondria-endoplasmic reticulum cortex anchor (MECA) is a well-known tethering complex in Saccharomyces cerevisiae that tethers mitochondria to the plasma membrane and plays a key role in mitochondrial fission. The main components of MECA are Num1 and Mdm36, and it is known that Mdm36 binds to Num1 to enhance mitochondrial tethering. To better understand the biochemical characteristics of the Num1-Mdm36 complex at the molecular level, we purified the coiled-coil domain of Num1, full-length Mdm36, and Num1-Mdm36 complex and identified the oligomeric state and stoichiometric characteristics of the Num1-Mdm36 complex by chemical crosslinking, size-exclusion chromatography coupled with multi-angle light scattering, and isothermal titration calorimetry. Mdm36 exists as a dimer and interacts preferentially with Num1 with a stoichiometry of 2:2, forming a heterotetrameric complex. Furthermore, we narrowed down the specific binding region of Num1, which is essential for interacting with Mdm36, and showed that their binding affinity is strong enough to tether both mitochondrial and plasma membranes. Our biochemical characterizations suggest a stoichiometric model of the Num1-Mdm36 complex at the mitochondria-plasma membrane contact site in budding yeast.

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Abstract : Phosphatidylserine (PS), a negatively charged phospholipid exclusively located in the inner leaflet of the plasma membrane, is involved in various cellular processes such as blood coagulation, myoblast fusion, mammalian fertilization, and clearance of apoptotic cells. Proteins that specifically interact with PS must be identified to comprehensively understand the cellular processes involving PS. However, only a limited number of proteins are known to associate with PS. To identify PS-associating proteins, we performed a pulldown assay using streptavidin-coated magnetic beads on which biotin-linked PS was immobilized. Using this approach, we identified Hsd17b4, a peroxisomal protein, as a PS-associating protein. Hsd17b4 strongly associated with PS, but not with phosphatidylcholine or sphingomyelin, and the Scp-2-like domain of Hsd17b4 was responsible for this association. The association was disrupted by PS in liposomes, but not by free PS or the components of PS. In addition, translocation of PS to the outer leaflet of the plasma membrane enriched Hsd17b4 in peroxisomes. Collectively, this study suggests an unexpected role of PS as a regulator of the subcellular localization of Hsd17b4.

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Abstract : Uniquely expressed in the colon, MS4A12 exhibits store-operated Ca²⁺ entry (SOCE) activity. However, compared to MS4A1 (CD20), a Ca²⁺ channel and ideal target for successful leukaemia immunotherapy, MS4A12 has rarely been studied. In this study, we investigated the involvement of MS4A12 in Ca²⁺ influx and expression changes in MS4A12 in human colonic malignancy. Fluorescence of GCaMP-fused MS4A12 (GCaMP-M12) was evaluated to analyse MS4A12 activity in Ca²⁺ influx. Plasma membrane expression of GCaMP-M12 was achieved by homo- or hetero-complex formation with no-tagged MS4A12 (nt-M12) or Orai1, respectively. GCaMP-M12 fluorescence in plasma membrane increased only after thapsigargin-induced depletion of endoplasmic reticulum Ca²⁺ stores, and this fluorescence was inhibited by typical SOCE inhibitors and siRNA for Orai1. Furthermore, GCaMP-MS4A12 and Orai1 co-transfection elicited greater plasma membrane fluorescence than GCaMP-M12 co-transfected with nt-M12. Interestingly, the fluorescence of GCaMP-M12 was decreased by STIM1 over-expression, while increased by siRNA for STIM1 in the presence of thapsigargin and extracellular Ca²⁺. Moreover, immunoprecipitation assay revealed that Orai1 co-expression decreased protein interactions between MS4A12 and STIM1. In human colon tissue, MS4A12 was expressed in the apical region of the colonic epithelium, although its expression was dramatically decreased in colon cancer tissues. In conclusion, we propose that MS4A12 contributes to SOCE through complex formation with Orai1, but does not cooperate with STIM1. Additionally, we discovered that MS4A12 is expressed in the apical membrane of the colonic epithelium and that its expression is decreased with cancer progression.

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Abstract : To define novel networks of Parkinson’s disease (PD) pathogenesis, the substantia nigra pars compacta of A53T mice, where a death-promoting protein, FAS-associated factor 1 was ectopically expressed for 2 weeks in the 2-, 4-, 6-, and 8-month-old mice, and was subjected to transcriptomic analysis. Compendia of expression profiles and a hierarchical clustering heat map of differentially expressed genes associated with PD were bioinformatically generated. Transcripts level of a particular gene was fluctuated by 20, 60, and 0.75 fold in the 4-, 6-, and 8-month-old mice compared to the 2 months old. Because the gene contained Kelch domain, it was named as Kapd (Kelch-containing protein associated with PD). Biological functions of Kapd were systematically investigated in the zebrafish embryos. First, transcripts of a zebrafish homologue of Kapd, kapd were found in the floor plate of the neural tube at 10 h post fertilization (hpf), and restricted to the tegmentum, hypothalamus, and cerebellum at 24 hpf. Second, knockdown of kapd caused developmental defects of DA progenitors in the midbrain neural keel and midbrain– hindbrain boundary at 10 hpf. Third, overexpression of kapd increased transcripts level of the dopaminergic immature neuron marker, shha in the prethalamus at 16.5 hpf. Finally, developmental consequences of kapd knockdown reduced transcripts level of the markers for the immature and mature DA neurons, nkx2.2, olig2, otx2b, and th in the ventral diencephalon of the midbrain at 18 hpf. It is thus most probable that Kapd play a key role in the specification of the DA neuronal precursors in zebrafish embryos.

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Abstract : Even though mesenchymal stem cells (MSCs) are known for cartilage regeneration, their therapeutic efficacy needs to be enhanced. In the present study, we produced genome-edited silent information regulator 2 type 1 (Sirt1)-overexpressing MSCs, and evaluated their therapeutic potential in a damaged cartilage mouse liver fibrosis model. The Sirt1 gene was successfully inserted into a ‘safe harbor’ genomic locus in amniotic mesenchymal stem cells (AMMs), and the chondrogenic properties of the Sirt1 gene overexpressing AMMs (AMM/S) were characterized using quantitative PCR and histology. Therapeutic potentials were investigated in a collagen-induced arthritis (CIA) mouse model. Chondrocyte-differentiated AMM/S expressed cartilage-specific genes and were positive for Safranin O staining. Transplantation of AMM/S attenuated CIA progression and suppressed T helper (Th)-17 cell activation while increasing the Treg cell population in CIA mice. Pro-inflammatory factors, such as interleukin (IL)-1β, IL-6, monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF)-α were significantly decreased in AMM/S-injected joint tissues. In conclusion, genome-edited AMM/S may represent a safe and alternative therapeutic option for the treatment and repair of damaged cartilage, or in inflammatory joint arthritis.

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Abstract : Numerous studies highlight the potential benefits potentials of supplemental cartilage oligomeric matrix protein-angiopoietin-1 (COMP-Ang1) through improved angiogenic effects. However, our recent findings show that excessive overexpression of COMP-Ang1 induces an impaired bone marrow (BM) microenvironment and senescence of hematopoietic stem cells (HSCs). Here, we investigated the underlying mechanisms of how excessive COMP-Ang1 affects the function of BM-conserved stem cells and hematopoiesis using K14-Cre;inducible-COMP-Ang1-transgenic mice. Excessive COMP-Ang1 induced peripheral egression and senescence of BM HSCs and mesenchymal stem cells (MSCs). Excessive COMP-Ang1 also caused abnormal hematopoiesis along with skewed differentiation of HSCs toward myeloid lineage rather than lymphoid lineage. Especially, excessive COMP-Ang1 disturbed late-stage erythroblast maturation, followed by decreased expression of stromal cell-derived factor 1 (SDF-1) and globin transcription factor 1 (GATA-1) and increased levels of superoxide anion and p-p38 kinase. However, transplantation with the mutant-derived BM cells or treatment with rhCOMP-Ang1 protein did not alter the frequency or GATA-1 expression of erythroblasts in recipient mice or in cultured BM cells. Together, our findings suggest that excessive COMP-Ang1 impairs the functions of BM HSCs and MSCs and hematopoietic processes, eventually leading to abnormal erythropoiesis via imbalanced SDF-1/CXCR4 axis and GATA-1 expression rather than Ang1/Tie2 signaling axis alterations.

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Abstract : Metallothionein (MT1M) belongs to a family of cysteine-rich cytosolic protein and has been reported to be a tumor suppressor gene in multiple cancers. However, its role in esophageal carcinoma carcinogenesis remains unclear. In this study, MT1M expression was correlated with tumor type, stage, drinking and smoking history, as well as patient survival. We also studied the regulation and biological function of MT1M in esophageal squamous cell carcinoma (ESCC). We have found that MT1M is significantly downregulated in ESCC tissues compared with adjacent non-cancer tissues. Furthermore, restoration of expression by treatment with the demethylation agent A + T showed that MT1M downregulation might be closely related to hypermethylation in its promoter region. Over-expression of MT1M in ESCC cells significantly altered cell morphology, induced apoptosis, and reduced colony formation, cell viability, migration and epithelial-mesenchymal transition. Moreover, based on reactive oxygen species (ROS) levels, a superoxide dismutase 1 (SOD1) activity assay and protein analysis, we verified that the tumor-suppressive function of MT1M was at least partially caused by its upregulation of ROS levels, downregulation of SOD1 activity and phosphorylation of the SOD1 downstream pathway PI3K/AKT. In conclusion, our results demonstrated that MT1M was a novel tumor-suppressor in ESCC and may be disrupted by promoter CpG methylation during esophageal carcinogenesis.