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European Journal of Cell Biology Jun 2024The Microphthalmia-associated Transcription Factor (MITF) governs numerous cellular and developmental processes. In mice, it promotes specification and differentiation...
The Microphthalmia-associated Transcription Factor (MITF) governs numerous cellular and developmental processes. In mice, it promotes specification and differentiation of the retinal pigmented epithelium (RPE), and in humans, some mutations in MITF induce congenital eye malformations. Herein, we explore the function and regulation of Mitf in Drosophila eye development and uncover two roles. We find that knockdown of Mitf results in retinal displacement (RDis), a phenotype associated with abnormal eye formation. Mitf functions in the peripodial epithelium (PE), a retinal support tissue akin to the RPE, to suppress RDis, via the Hippo pathway effector Yorkie (Yki). Yki physically interacts with Mitf and can modify its transcriptional activity in vitro. Severe loss of Mitf, instead, results in the de-repression of retinogenesis in the PE, precluding its development. This activity of Mitf requires the protein phosphatase 2 A holoenzyme STRIPAK-PP2A, but not Yki; Mitf transcriptional activity is potentiated by STRIPAK-PP2A in vitro and in vivo. Knockdown of STRIPAK-PP2A results in cytoplasmic retention of Mitf in vivo and in its decreased stability in vitro, highlighting two potential mechanisms for the control of Mitf function by STRIPAK-PP2A. Thus, Mitf functions in a context-dependent manner as a key determinant of form and fate in the Drosophila eye progenitor epithelium.
Topics: Animals; Drosophila Proteins; Microphthalmia-Associated Transcription Factor; YAP-Signaling Proteins; Trans-Activators; Nuclear Proteins; Eye; Protein Phosphatase 2; Drosophila melanogaster; Epithelium; Cell Differentiation; Homeodomain Proteins
PubMed: 38776620
DOI: 10.1016/j.ejcb.2024.151421 -
Proceedings of the National Academy of... May 2024C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) is a noncanonical protein serine/threonine phosphatase that has a conserved role in regulating ER membrane...
C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) is a noncanonical protein serine/threonine phosphatase that has a conserved role in regulating ER membrane biogenesis. Inactivating mutations in CTDNEP1 correlate with the development of medulloblastoma, an aggressive childhood cancer. The transmembrane protein Nuclear Envelope Phosphatase 1 Regulatory Subunit 1 (NEP1R1) binds CTDNEP1, but the molecular details by which NEP1R1 regulates CTDNEP1 function are unclear. Here, we find that knockdown of NEP1R1 generates identical phenotypes to reported loss of CTDNEP1 in mammalian cells, establishing CTDNEP1-NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. Mechanistically, NEP1R1 acts as an activating regulatory subunit that directly binds and increases the phosphatase activity of CTDNEP1. By defining a minimal NEP1R1 domain sufficient to activate CTDNEP1, we determine high-resolution crystal structures of the CTDNEP1-NEP1R1 complex bound to a peptide sequence acting as a pseudosubstrate. Structurally, NEP1R1 engages CTDNEP1 at a site distant from the active site to stabilize and allosterically activate CTDNEP1. Substrate recognition is facilitated by a conserved Arg residue in CTDNEP1 that binds and orients the substrate peptide in the active site. Together, this reveals mechanisms for how NEP1R1 regulates CTDNEP1 and explains how cancer-associated mutations inactivate CTDNEP1.
Topics: Humans; Crystallography, X-Ray; Endoplasmic Reticulum; Intracellular Membranes; Membrane Proteins; Phosphoprotein Phosphatases; Protein Binding
PubMed: 38776370
DOI: 10.1073/pnas.2321167121 -
Journal of Agricultural and Food... Jun 2024Global water deficit is a severe abiotic stress threatening the yielding and quality of crops. Abscisic acid (ABA) is a phytohormone that mediates drought tolerance....
Global water deficit is a severe abiotic stress threatening the yielding and quality of crops. Abscisic acid (ABA) is a phytohormone that mediates drought tolerance. Protein kinases and phosphatases function as molecular switches in eukaryotes. Protein phosphatases type 2C (PP2Cs) are a major family that play essential roles in ABA signaling and stress responses. However, the role and underlying mechanism of PP2C in rapeseed ( L.) mediating drought response has not been reported yet. Here, we characterized a PP2C family member, BnaPP2C37, and its expression level was highly induced by ABA and dehydration treatments. It negatively regulates drought tolerance in rapeseed. We further identified that BnaPP2C37 interacted with multiple PYR/PYL receptors and a drought regulator BnaCPK5 (calcium-dependent protein kinase 5) through yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Specifically, BnaPYL1 and BnaPYL9 repress BnaPP2C37 phosphatase activity. Moreover, the pull-down assay and phosphatase assays show BnaPP2C37 interacts with BnaCPK5 to dephosphorylate BnaCPK5 and its downstream BnaABF3. Furthermore, a dual-luciferase assay revealed transcript level was enhanced by BnaABF3 and BnaABF4, forming a negative feedback regulation to ABA response. In summary, we identified that BnaPP2C37 functions negatively in drought tolerance of rapeseed, and its phosphatase activity is repressed by BnaPYL1/9 whereas its transcriptional level is upregulated by BnaABF3/4.
Topics: Abscisic Acid; Plant Proteins; Brassica napus; Droughts; Gene Expression Regulation, Plant; Protein Phosphatase 2C; Stress, Physiological; Plant Growth Regulators; Phosphoprotein Phosphatases; Drought Resistance
PubMed: 38771652
DOI: 10.1021/acs.jafc.4c00385 -
Molecular Pharmacology Jun 2024Remdesivir (RDV), a broad-spectrum antiviral agent, is often used together with dexamethasone (DEX) for hospitalized COVID-19 patients requiring respiratory support....
Remdesivir (RDV), a broad-spectrum antiviral agent, is often used together with dexamethasone (DEX) for hospitalized COVID-19 patients requiring respiratory support. Potential hepatic adverse drug reaction is a safety concern associated with the use of RDV. We previously reported that DEX cotreatment effectively mitigates RDV-induced hepatotoxicity and reduces elevated serum alanine aminotransferase and aspartate aminotransferase levels in cultured human primary hepatocytes (HPH) and hospitalized COVID-19 patients, respectively. Yet, the precise mechanism behind this protective drug-drug interaction remains largely unknown. Here, we show that through the activation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling, RDV induces apoptosis (cleavage of caspases 8, 9, and 3), autophagy (increased autophagosome and LC3-II), and mitochondrial damages (decreased membrane potential, respiration, ATP levels, and increased expression of Bax and the released cytosolic cytochrome C) in HPH. Importantly, cotreatment with DEX partially reversed RDV-induced apoptosis, autophagy, and cell death. Mechanistically, DEX deactivates/dephosphorylates p38, JNK, and ERK1/2 signaling by enhancing the expression of dual specificity protein phosphatase 1 (DUSP1), a mitogen-activated protein kinase (MAPK) phosphatase, in a glucocorticoid receptor (GR)-dependent manner. Knockdown of GR in HPH attenuates DEX-mediated DUSP1 induction, MAPK dephosphorylation, as well as protection against RDV-induced hepatotoxicity. Collectively, our findings suggest a molecular mechanism by which DEX modulates the GR-DUSP1-MAPK regulatory axis to alleviate the adverse actions of RDV in the liver. SIGNIFICANCE STATEMENT: The research uncovers the molecular mechanisms by which dexamethasone safeguards against remdesivir-associated liver damage in the context of COVID-19 treatment.
Topics: Dexamethasone; Humans; Adenosine Monophosphate; Alanine; Chemical and Drug Induced Liver Injury; Antiviral Agents; COVID-19 Drug Treatment; Dual Specificity Phosphatase 1; Hepatocytes; Apoptosis; Autophagy; Cells, Cultured; MAP Kinase Signaling System
PubMed: 38769019
DOI: 10.1124/molpharm.124.000894 -
Journal of Immunology (Baltimore, Md. :... Jul 2024The JAK-STAT pathway is a central communication node for various biological processes. Its activation is characterized by phosphorylation and nuclear translocation of...
The JAK-STAT pathway is a central communication node for various biological processes. Its activation is characterized by phosphorylation and nuclear translocation of the transcription factor STAT. The regulatory balance of JAK-STAT signaling is important for maintenance of immune homeostasis. Protein tyrosine phosphatases (PTPs) induce dephosphorylation of tyrosine residues in intracellular proteins and generally function as negative regulators in cell signaling. However, the roles of PTPs in JAK-STAT signaling, especially in invertebrates, remain largely unknown. Pacific white shrimp Penaeus vannamei is currently an important model for studying invertebrate immunity. This study identified a novel member of the dual-specificity phosphatase (DUSP) subclass of the PTP superfamily in P. vannamei, named PvDUSP14. By interacting with and dephosphorylating STAT, PvDUSP14 inhibits the excessive activation of the JAK-STAT pathway, and silencing of PvDUSP14 significantly enhances humoral and cellular immunity in shrimp. The promoter of PvDUSP14 contains a STAT-binding motif and can be directly activated by STAT, suggesting that PvDUSP14 is a regulatory target gene of the JAK-STAT pathway and mediates a negative feedback regulatory loop. This feedback loop plays a role in maintaining homeostasis of JAK-STAT signaling and is involved in antibacterial and antiviral immune responses in shrimp. Therefore, the current study revealed a novel inhibitory mechanism of JAK-STAT signaling, which is of significance for studying the regulatory mechanisms of immune homeostasis in invertebrates.
Topics: Animals; Penaeidae; Signal Transduction; Janus Kinases; STAT Transcription Factors; Feedback, Physiological; Phosphorylation; Dual-Specificity Phosphatases; Arthropod Proteins
PubMed: 38767414
DOI: 10.4049/jimmunol.2300871 -
Stem Cell Research Jun 2024Spinocerebellar ataxia type 12 (SCA12) is caused by a CAG expansion mutation in PPP2R2B, a gene encoding brain-specific regulatory units of protein phosphatase 2A...
Spinocerebellar ataxia type 12 (SCA12) is caused by a CAG expansion mutation in PPP2R2B, a gene encoding brain-specific regulatory units of protein phosphatase 2A (PP2A); while normal alleles carry 4 to 31 triplets, the disease alleles carry 43 to 78 triplets. Here, by CRISPR/Cas9n genome editing, we have generated a human heterozygous SCA12 iPSC line with 73 triplets for the mutant allele. The heterozygous SCA12 iPSCs have normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.
Topics: Humans; Induced Pluripotent Stem Cells; Gene Editing; Spinocerebellar Ataxias; Heterozygote; Mutation; Cell Line; CRISPR-Cas Systems; Protein Phosphatase 2; Nerve Tissue Proteins
PubMed: 38759410
DOI: 10.1016/j.scr.2024.103441 -
International Immunopharmacology Jun 2024Alzheimer's disease (AD) is a progressive neurodegenerative disease. Neuronal calcium overload plays an important role in Aβ deposition and neuroinflammation, which are...
Alzheimer's disease (AD) is a progressive neurodegenerative disease. Neuronal calcium overload plays an important role in Aβ deposition and neuroinflammation, which are strongly associated with AD. However, the specific mechanisms by which calcium overload contributes to neuroinflammation and AD and the relationship between them have not been elucidated. Phospholipase C (PLC) is involved in regulation of calcium homeostasis, and CN-NFAT1 signaling is dependent on intracellular Ca ([Ca]) to regulate transcription of genes. Therefore, we hypothesized that the PLC-CN-NFAT1 signaling might mediate the interaction between Aβ and inflammation to promote neuronal injury in AD. In this experiment, the results showed that the levels of Aβ, IL-1β and [Ca] in the hippocampal primary neurons of APP/PS1 mice (APP neurons) were significantly increased. IL-1β exposure also significantly increased Aβ and [Ca] in HT22 cells, suggesting a close association between Aβ and IL-1β in the development of AD. Furthermore, PLC activation induced significant calcium homeostasis imbalance, cell apoptosis, Aβ and ROS production, and significantly increased expressions of CN and NFAT1, while PLC inhibitor significantly reversed these changes in APP neurons and IL-1β-induced HT22 cells. Further results indicated that PLC activation significantly increased the expressions of NOX2, APP, BACE1, and NCSTN, which were inhibited by PLC inhibitor in APP neurons and IL-1β-induced HT22 cells. All indications point to a synergistic interaction between Aβ and IL-1β by activating the PLC-CN-NFAT1 signal, ultimately causing a vicious cycle, resulting in neuronal damage in AD. The study may provide a new idea and target for treatment of AD.
Topics: Animals; Hippocampus; Interleukin-1beta; Neurons; NFATC Transcription Factors; Mice; Type C Phospholipases; Alzheimer Disease; Signal Transduction; Amyloid beta-Peptides; Calcineurin; Mice, Transgenic; Calcium; Cell Line; Humans; Cells, Cultured; Apoptosis; Reactive Oxygen Species; Mice, Inbred C57BL; Amyloid beta-Protein Precursor
PubMed: 38749336
DOI: 10.1016/j.intimp.2024.112259 -
Biochemical and Biophysical Research... Jul 2024Osteoclasts are multinucleated cells with bone resorption activity. Excessive osteoclast activity has been implicated in osteoporosis, rheumatoid arthritis, and bone...
Osteoclasts are multinucleated cells with bone resorption activity. Excessive osteoclast activity has been implicated in osteoporosis, rheumatoid arthritis, and bone destruction due to bone metastases from cancer, making osteoclasts essential target cells in bone and joint diseases. C-terminal domain nuclear envelope phosphatase 1 (Ctdnep1, formerly Dullard) is a negative regulator of transforming growth factor (TGF)-β superfamily signaling and regulates endochondral ossification in mesenchymal cells during skeletal development. In this study, we investigated the role of Ctdnep1 in the Receptor activator of nuclear factor-kappa B ligand (RANKL)-induced RAW264.7 osteoclast differentiation. Expression of Ctdnep1 did not change during osteoclast differentiation; Ctdnep1 protein localized to the cytoplasm before and after osteoclast differentiation. Small interfering RNA-mediated knockdown of Ctdnep1 increased tartrate-resistant acid phosphatase-positive multinucleated osteoclasts and the expression of osteoclast marker genes, including Acp5, Ctsk, and Nfatc1. Interestingly, the knockdown of Ctdnep1 increased the protein level of Nfatc1 in cells unstimulated with RANKL. Knockdown of Ctdnep1 also enhanced calcium-resorbing activity. Mechanistically, the knockdown of Ctdnep1 increased the phosphorylation of RANKL signaling components. These results suggest that Ctdnep1 negatively regulates osteoclast differentiation by suppressing the RANKL signaling pathway.
Topics: Animals; Mice; Cell Differentiation; Gene Knockdown Techniques; NFATC Transcription Factors; Osteoclasts; RANK Ligand; RAW 264.7 Cells; Phosphoprotein Phosphatases
PubMed: 38749090
DOI: 10.1016/j.bbrc.2024.150063 -
Journal of Applied Oral Science :... 2024Studies have highlighted numerous benefits of ozone therapy in the field of medicine and dentistry, including its antimicrobial efficacy against various pathogenic...
OBJECTIVE
Studies have highlighted numerous benefits of ozone therapy in the field of medicine and dentistry, including its antimicrobial efficacy against various pathogenic microorganisms, its ability to modulate the immune system effectively, reduce inflammation, prevent hypoxia, and support tissue regeneration. However, its effects on dental extraction healing remain to be elucidated. .Therefore, this study aimed to evaluate the effects of systemically administered ozone (O3) at different doses in the healing of dental extraction sockets in rats.
METHODOLOGY
To this end, 72 Wistar rats were randomly divided into four groups after extraction of the right upper central incisor: Group C - control, no systemic treatment; Group OZ0.3 - animals received a single dose of 0.3 mg/kg O3; Group OZ0.7 - a single dose of 0.7 mg/kg O3; and Group OZ1.0 - a single dose of 1.0 mg/kg O3, intraperitoneally. In total, six animals from each group were euthanized at 7, 14, and 21 days after the commencement of treatment. Bone samples were harvested and further analyzed by descriptive histology, histomorphometry, and immunohistochemistry for osteocalcin (OCN) and tartrate-resistant acid phosphatase (TRAP) protein expression.
RESULTS
All applied doses of O3 were shown to increase the percentage of bone tissue (PBT) after 21 days compared to group C. After 14 days, the OZ0.7 and OZ1.0 groups showed significantly higher PBT when compared to group C. The OZ1.0 group presented the most beneficial results regarding PBT among groups, which denotes a dose-dependent response. OCN immunostaining was higher in all groups at 21 days. However, after seven and 14 days, the OZ1.0 group showed a significant increase in OCN immunostaining compared to C group. No differences in TRAP+ osteoclasts were found between groups and time points.
CONCLUSION
Therefore, O3 therapy at higher doses might be beneficial for bone repair of the alveolar socket following tooth extraction.
Topics: Animals; Ozone; Rats, Wistar; Tooth Extraction; Tooth Socket; Wound Healing; Tartrate-Resistant Acid Phosphatase; Immunohistochemistry; Osteocalcin; Time Factors; Male; Random Allocation; Reproducibility of Results; Treatment Outcome; Reference Values
PubMed: 38747807
DOI: 10.1590/1678-7757-2023-0412 -
Acta Physiologica (Oxford, England) Jul 2024With a focus on the discrepancy between preclinical and clinical findings, this review will gather comprehensive information about the effects of calcineurin inhibitors... (Review)
Review
AIMS
With a focus on the discrepancy between preclinical and clinical findings, this review will gather comprehensive information about the effects of calcineurin inhibitors (CNI) on cognitive function and related brain pathology from in vitro, in vivo, and clinical studies. We also summarize the potential mechanisms that underlie the pathways related to CNI-induced cognitive impairment.
METHODS
We systematically searched articles in PubMed using keywords 'calcineurin inhibitor*' and 'cognition' to identify related articles, which the final list pertaining to underlying mechanisms of CNI on cognition.
RESULTS
Several studies have reported an association between calcineurin and the neuropathology of Alzheimer's disease (AD). AD is the most common neurocognitive disorder associated with amyloid plaques and neurofibrillary tangles in the brain, leading to cognitive impairment. CNI, including tacrolimus and cyclosporin A, are commonly prescribed for patients with transplantation of solid organs such as kidney, liver, or heart, those drugs are currently being used as long-term immunosuppressive therapy. Although preclinical models emphasize the favorable effects of CNI on the restoration of brain pathology due to the impacts of calcineurin on the alleviation of amyloid-beta deposition and tau hyperphosphorylation, or rescuing synaptic and mitochondrial functions, treatment-related neurotoxicity, resulting in cognitive dysfunctions has been observed in clinical settings of patients who received CNI.
CONCLUSION
Inconsistent results of CNI on cognition from clinical studies have been observed due to impairment of the blood-brain barrier, neuroinflammation mediated by reactive oxygen species, and alteration in mitochondrial fission, and extended research is required to confirm its promising use in cognitive impairment.
Topics: Humans; Calcineurin Inhibitors; Animals; Cognition; Cognitive Dysfunction; Brain; Calcineurin; Alzheimer Disease; Immunosuppressive Agents
PubMed: 38747643
DOI: 10.1111/apha.14161