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Acta Neuropathologica Communications May 2024Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies....
Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-min and 1-h postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson's disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.
Topics: Animals; Humans; Male; Mice; Alkaline Phosphatase; alpha-Synuclein; Brain; Mice, Inbred C57BL; Mice, Transgenic; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Aggregates; Protein Aggregation, Pathological; Synucleinopathies
PubMed: 38822421
DOI: 10.1186/s40478-024-01785-0 -
Nature Communications May 2024The two-pore domain potassium (K) channels TREK-1 and TREK-2 link neuronal excitability to a variety of stimuli including mechanical force, lipids, temperature and...
The two-pore domain potassium (K) channels TREK-1 and TREK-2 link neuronal excitability to a variety of stimuli including mechanical force, lipids, temperature and phosphorylation. This regulation involves the C-terminus as a polymodal stimulus sensor and the selectivity filter (SF) as channel gate. Using crystallographic up- and down-state structures of TREK-2 as a template for full atomistic molecular dynamics (MD) simulations, we reveal that the SF in down-state undergoes inactivation via conformational changes, while the up-state structure maintains a stable and conductive SF. This suggests an atomistic mechanism for the low channel activity previously assigned to the down state, but not evident from the crystal structure. Furthermore, experimentally by using (de-)phosphorylation mimics and chemically attaching lipid tethers to the proximal C-terminus (pCt), we confirm the hypothesis that moving the pCt towards the membrane induces the up-state. Based on MD simulations, we propose two gating pathways by which movement of the pCt controls the stability (i.e., conductivity) of the filter gate. Together, these findings provide atomistic insights into the SF gating mechanism and the physiological regulation of TREK channels by phosphorylation.
Topics: Potassium Channels, Tandem Pore Domain; Molecular Dynamics Simulation; Humans; Phosphorylation; Ion Channel Gating; Protein Domains; Cytosol; Animals; HEK293 Cells; Crystallography, X-Ray
PubMed: 38821927
DOI: 10.1038/s41467-024-48823-y -
Neuroreport Jul 2024Hyperactivation of the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) is observed in reactive astrocytes associated with neuroinflammation and progressive...
Hyperactivation of the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) is observed in reactive astrocytes associated with neuroinflammation and progressive degenerative diseases, like Alzheimer's disease. Apart from key transcription factors (e.g. nuclear factor of activated t cells and nuclear factor-κB) very few other CN-dependent pathways have been studied in astrocytes. The hemichannel protein, connexin 43 (Cx43) is found at high levels in astrocytes and contains a CN-sensitive Ser residue near its carboxy terminus. CN-dependent dephosphorylation of Cx43 has been reported in primary astrocytes treated with injurious stimuli, but much remains unknown about CN/Cx43 interactions in the context of neuroinflammation and disease. Western blots were used to assess total Cx43 and dephosphorylated Cx43 subtypes in rat embryonic primary astrocytes treated with a hyperactive CN fragment (ΔCN, via adenovirus), or with a proinflammatory cytokine cocktail. Under similar treatment conditions, an ethidium bromide (EtBr) uptake assay was used to assess membrane permeability. Effects of ΔCN and cytokines were tested in the presence or absence of the CN inhibitor, cyclosporin A. A connexin inhibitor, carbenoxolone was also used in EtBr assays to assess the involvement of connexins in membrane permeability. Treatment with ΔCN or cytokines increased dephosphorylated Cx43 levels in conjunction with increased membrane permeability (elevated EtBr uptake). Effects of ΔCN or cytokine treatment were blocked by cyclosporine A. Treatment-induced changes in EtBr uptake were also inhibited by carbenoxolone. The results suggest that Cx43 hemichannels could be an important mechanism through which astrocytic CN disrupts neurologic function associated with neurodegenerative disease.
Topics: Astrocytes; Connexin 43; Animals; Phosphorylation; Calcineurin; Rats; Cell Membrane Permeability; Cells, Cultured; Rats, Sprague-Dawley
PubMed: 38813906
DOI: 10.1097/WNR.0000000000002051 -
Zhongguo Zhong Yao Za Zhi = Zhongguo... May 2024Sequential catalysis by ent-copalyl diphosphate(CPS) and ent-kaurene synthase(KS) is a critical step for plants to initiate the biosynthesis of gibberellin with...
Sequential catalysis by ent-copalyl diphosphate(CPS) and ent-kaurene synthase(KS) is a critical step for plants to initiate the biosynthesis of gibberellin with geranylgeranyl pyrophosphate(GGPP) as the substrate. This study mined the transcriptome data of Stellera chamaejasme and cloned two key diterpene synthase genes, SchCPS and SchKS, involved in the gibberellin pathway. The two genes had the complete open reading frames of 2 595 bp and 1 701 bp, encoding two hydrophilic proteins composed of 864 and 566 amino acid residues and with the relative molecular mass of 97.9 kDa and 64.6 kDa and the theoretical isoelectric points of 5.61 and 6.12, respectively. Sequence comparison and phylogenetic tree showed that SchCPS contained LHS, PNV, and DxDD motifs conserved in the CPS family and was categorized in the TPS-c subfamily, while SchKS contained DDxxD, NSE/DTE and PIx motifs conserved in the KS family and was categorized in the TPS-e subfamily. Functional validation showed that SchCPS catalyzed the protonation and cyclization of GGPP to ent-CPP, while SchKS acted on ent-CPP dephosphorylation and re-cyclization to ent-kaurene. In this study, the full-length sequences of SchCPS and SchKS were cloned and functionally verified for the first time, which not only enriched the existing CPS and KS gene libraries but also laid a foundation for the cloning and biosynthesis pathway analysis of more genes involved in the synthesis of active components in S. chamaejasme.
Topics: Alkyl and Aryl Transferases; Phylogeny; Thymelaeaceae; Plant Proteins; Amino Acid Sequence; Diterpenes, Kaurane; Sequence Alignment; Cloning, Molecular
PubMed: 38812142
DOI: 10.19540/j.cnki.cjcmm.20240214.102 -
Journal of Medicinal Chemistry Jun 2024High extracellular concentrations of adenosine triphosphate (ATP) in the tumor microenvironment generate adenosine by sequential dephosphorylation of CD39 and CD73,...
High extracellular concentrations of adenosine triphosphate (ATP) in the tumor microenvironment generate adenosine by sequential dephosphorylation of CD39 and CD73, resulting in potent immunosuppression to inhibit T cell and natural killer (NK) cell function. CD73, as the determining enzyme for adenosine production, has been shown to correlate with poor clinical tumor prognosis. Conventional inhibitors as analogues of adenosine 5'-monophosphate (AMP) may have a risk of further metabolism to adenosine analogues. Here, we report a new series of malonic acid non-nucleoside inhibitors coordinating with zinc ions of CD73. Compound was found to be a superior CD73 inhibitor (IC = 60 nM) by structural optimization, and its pharmacokinetic properties were investigated. In mouse tumor models, compound showed excellent efficacy and reversal of immunosuppression in combination with chemotherapeutic agents or checkpoint inhibitors, suggesting that it deserves further development as a novel CD73 inhibitor.
Topics: 5'-Nucleotidase; Animals; Humans; Mice; Malonates; Zinc; Structure-Activity Relationship; Enzyme Inhibitors; GPI-Linked Proteins; Antineoplastic Agents; Drug Discovery; Cell Line, Tumor
PubMed: 38809692
DOI: 10.1021/acs.jmedchem.4c00825 -
Aging May 2024Glioblastoma multiforme (GBM) is the most prevalent and lethal primary intracranial neoplasm in the adult population, with treatments of limited efficacy. Recently,...
Glioblastoma multiforme (GBM) is the most prevalent and lethal primary intracranial neoplasm in the adult population, with treatments of limited efficacy. Recently, bufotalin has been shown to have anti-cancer activity in a variety of cancers. This investigation aims to investigate the effect of bufotalin on GBM and elucidate its potential underlying mechanism. Our results show that bufotalin not only inhibits the proliferation and epithelial-mesenchymal transition (EMT) but also triggers apoptosis in GBM cells. The result of RNA-seq indicated that bufotalin could induce mitochondrial dysfunction. Moreover, our observations indicate that bufotalin induces an excessive accumulation of intracellular reactive oxygen species (ROS) in GBM cells, leading to mitochondrial dysfunction and the dephosphorylation of AKT. Moreover, bufotalin improved TMZ sensitivity of GBM cells and . In conclusion, bufotalin enhances apoptosis and TMZ chemosensitivity of glioblastoma cells by promoting mitochondrial dysfunction via AKT signaling pathway.
Topics: Glioblastoma; Humans; Apoptosis; Mitochondria; Proto-Oncogene Proteins c-akt; Signal Transduction; Bufanolides; Cell Line, Tumor; Animals; Reactive Oxygen Species; Cell Proliferation; Mice; Brain Neoplasms; Epithelial-Mesenchymal Transition
PubMed: 38809514
DOI: 10.18632/aging.205883 -
Journal of Bacteriology Jun 2024The EnvZ-OmpR two-component system of regulates the expression of the and porin genes in response to medium osmolarity. However, certain mutations in confer...
The EnvZ-OmpR two-component system of regulates the expression of the and porin genes in response to medium osmolarity. However, certain mutations in confer pleiotropy by affecting the expression of genes of the iron and maltose regulons not normally controlled by EnvZ-OmpR. In this study, we obtained two novel and pleiotropic alleles, and , among revertants of a mutant with heightened envelope stress and an outer membrane (OM) permeability defect. Unlike , pleiotropic mutations in have not been described previously. The mutant alleles reduced the expression of several outer membrane proteins (OMPs), overcame the temperature-sensitive growth defect of a protease-deficient (Δ) strain, and lowered envelope stress and OM permeability defects in a background lacking the BamB protein of an essential β-barrel assembly machinery complex. Biochemical analysis showed OmpRL19Q, like wild-type OmpR, is readily phosphorylated by EnvZ, but the EnvZ-dependent dephosphorylation of OmpRL19Q~P was drastically impaired compared to wild-type OmpR. This defect would lead to a prolonged half-life for OmpRL19Q~P, an outcome remarkably similar to what we had previously described for EnvZR397L, resulting in pleiotropy. By employing null alleles of the OMP genes, it was determined that the three pleiotropic alleles lowered envelope stress by reducing OmpF and LamB levels. The absence of LamB was principally responsible for lowering the OM permeability defect, as assessed by the reduced sensitivity of a Δ mutant to vancomycin and rifampin. Possible mechanisms by which novel EnvZ and OmpR mutants influence EnvZ-OmpR interactions and activities are discussed.IMPORTANCEMaintenance of the outer membrane (OM) integrity is critical for the survival of Gram-negative bacteria. Several envelope homeostasis systems are activated when OM integrity is perturbed. Through the isolation and characterization of novel pleiotropic / alleles, this study highlights the involvement of the EnvZ-OmpR two-component system in lowering envelope stress and the OM permeability defect caused by the loss of proteins that are involved in OM biogenesis, envelope homeostasis, and structural integrity.
Topics: Escherichia coli Proteins; Escherichia coli; Gene Expression Regulation, Bacterial; Bacterial Outer Membrane Proteins; Anti-Bacterial Agents; Alleles; Bacterial Proteins; Porins; Mutation; Stress, Physiological; Phosphorylation; Multienzyme Complexes; Trans-Activators
PubMed: 38809006
DOI: 10.1128/jb.00172-24 -
Frontiers in Immunology 2024In classical Hodgkin lymphoma (cHL), the survival of neoplastic cells is mediated by the activation of NF-κB, JAK/STAT and PI3K/Akt signaling pathways. CK2 is a highly...
INTRODUCTION
In classical Hodgkin lymphoma (cHL), the survival of neoplastic cells is mediated by the activation of NF-κB, JAK/STAT and PI3K/Akt signaling pathways. CK2 is a highly conserved serine/threonine kinase, consisting of two catalytic (α) and two regulatory (β) subunits, which is involved in several cellular processes and both subunits were found overexpressed in solid tumors and hematologic malignancies.
METHODS AND RESULTS
Biochemical analyses and assays showed an impaired expression of CK2 subunits in cHL, with CK2α being overexpressed and a decreased expression of CK2β compared to normal B lymphocytes. Mechanistically, CK2β was found to be ubiquitinated in all HL cell lines and consequently degraded by the proteasome pathway. Furthermore, at basal condition STAT3, NF-kB and AKT are phosphorylated in CK2-related targets, resulting in constitutive pathways activation. The inhibition of CK2 with CX-4945/silmitasertib triggered the de-phosphorylation of NF-κB-S529, STAT3-S727, AKT-S129 and -S473, leading to cHL cell lines apoptosis. Moreover, CX-4945/silmitasertib was able to decrease the expression of the immuno-checkpoint CD274/PD-L1 but not of CD30, and to synergize with monomethyl auristatin E (MMAE), the microtubule inhibitor of brentuximab vedotin.
CONCLUSIONS
Our data point out a pivotal role of CK2 in the survival and the activation of key signaling pathways in cHL. The skewed expression between CK2α and CK2β has never been reported in other lymphomas and might be specific for cHL. The effects of CK2 inhibition on PD-L1 expression and the synergistic combination of CX-4945/silmitasertib with MMAE pinpoints CK2 as a high-impact target for the development of new therapies for cHL.
Topics: Humans; Hodgkin Disease; Casein Kinase II; Signal Transduction; B7-H1 Antigen; Cell Line, Tumor; Phenazines; Naphthyridines; Apoptosis; Gene Expression Regulation, Neoplastic; Phosphorylation
PubMed: 38807597
DOI: 10.3389/fimmu.2024.1393485 -
Open Biology May 2024The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The...
The precise spatial and temporal control of histone phosphorylations is important for the ordered progression through the different phases of mitosis. The phosphorylation of H2B at S6 (H2B S6ph), which is crucial for chromosome segregation, reaches its maximum level during metaphase and is limited to the inner centromere. We discovered that the temporal and spatial regulation of this modification, as well as its intensity, are governed by the scaffold protein RepoMan and its associated catalytically active phosphatases, PP1α and PP1γ. Phosphatase activity is inhibited at the area of maximal H2B S6 phosphorylation at the inner centromere by site-specific Aurora B-mediated inactivation of the PP1/RepoMan complex. The motor protein Mklp2 contributes to the relocalization of Aurora B from chromatin to the mitotic spindle during anaphase, thus alleviating Aurora B-dependent repression of the PP1/RepoMan complex and enabling dephosphorylation of H2B S6. Accordingly, dysregulation of Mklp2 levels, as commonly observed in tumour cells, leads to the lack of H2B S6 dephosphorylation during early anaphase, which might contribute to chromosomal instability.
Topics: Aurora Kinase B; Phosphorylation; Humans; Histones; Mitosis; Protein Phosphatase 1; Cell Cycle Proteins; HeLa Cells; Spindle Apparatus; Centromere; Nuclear Proteins
PubMed: 38806145
DOI: 10.1098/rsob.230460 -
Biophysics and Physicobiology 2024KaiC is a multifunctional enzyme functioning as the core of the circadian clock system in cyanobacteria: its N-terminal domain has adenosine triphosphatase (ATPase)...
KaiC is a multifunctional enzyme functioning as the core of the circadian clock system in cyanobacteria: its N-terminal domain has adenosine triphosphatase (ATPase) activity, and its C-terminal domain has autokinase and autophosphatase activities targeting own S431 and T432. The coordination of these multiple biochemical activities is the molecular basis for robust circadian rhythmicity. Therefore, much effort has been devoted to elucidating the cooperative relationship between the two domains. However, structural and functional relationships between the two domains remain unclear especially with respect to the dawn phase, at which KaiC relieves its nocturnal history through autodephosphorylation. In this study, we attempted to design a double mutation of S431 and T432 that can capture KaiC as a fully dephosphorylated form with minimal impacts on its structure and function, and investigated the cooperative relationship between the two domains in the night to morning phases from many perspectives. The results revealed that both domains cooperate at the dawn phase through salt bridges formed between the domains, thereby non-locally co-activating two events, ATPase de-inhibition and S431 dephosphorylation. Our further analysis using existing crystal structures of KaiC suggests that the states of both domains are not always in one-to-one correspondence at every phase of the circadian cycle, and their coupling is affected by the interactions with KaiA or adjacent subunits within a KaiC hexamer.
PubMed: 38803331
DOI: 10.2142/biophysico.bppb-v21.0001