Characterization of a κ-Carrageenase from Marine Cellulophaga lytica strain N5-2 and Analysis of Its Degradation Products

A carrageenan-degrading marine Cellulophaga lytica strain N5-2 was isolated from the sediment of carrageenan production base. A κ-carrageenase (EC 3.2.1.83) with high activity was purified to electrophoretic homogeneity from the culture supernatant by a procedure of ammonium sulfate precipitation, dialyzing and gel filtration on SephadexG-200 and SephadexG-75. The purified enzyme was verified as a single protein on SDS-PAGE, and whose molecular weight was 40.8 kDa. The κ-carrageenase yielded a high activity of 1170 U/mg protein. For κ-carrageenase activity, the optimum temperature and pH were 35 °C and pH 7.0, respectively. The enzyme was stable at 40 °C for at least 2.5 h. The enzyme against κ-carrageenan gave a K_m value of 1.647 mg/mL and a V_max value of 8.7 μmol/min/mg when the reaction was carried out at 35 °C and pH 7.0. The degradation products of the κ-carrageenase were analyzed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), electrospray ionization time-of-flight mass spectroscopy (ESI-TOF-MS) and ¹³C-NMR spectroscopy, and the results indicated that the enzyme was specific of the β-1,4 linkage and hydrolyzed κ-carrageenan into κ-neocarraoctaose-sulfate and κ-neocarrahexaose-sulfate first, and then broke κ-neocarraoctaose-sulfate into κ-neocarrabiose-sulfate and κ-neocarrahexaose-sulfate.     

Interferon Beta Activity Is Modulated via Binding of Specific S100 Proteins

Interferon-β (IFN-β) is a pleiotropic cytokine used for therapy of multiple sclerosis, which is also effective in suppression of viral and bacterial infections and cancer. Recently, we reported a highly specific interaction between IFN-β and S100P lowering IFN-β cytotoxicity to cancer cells (Int J Biol Macromol. 2020; 143: 633–639). S100P is a member of large family of multifunctional Ca²⁺-binding proteins with cytokine-like activities. To probe selectivity of IFN-β—S100 interaction with respect to S100 proteins, we used surface plasmon resonance spectroscopy, chemical crosslinking, and crystal violet assay. Among the thirteen S100 proteins studied S100A1, S100A4, and S100A6 proteins exhibit strictly Ca²⁺-dependent binding to IFN-β with equilibrium dissociation constants, K_d, of 0.04–1.5 µM for their Ca²⁺-bound homodimeric forms. Calcium depletion abolishes the S100—IFN-β interactions. Monomerization of S100A1/A4/A6 decreases K_d values down to 0.11–1.0 nM. Interferon-α is unable of binding to the S100 proteins studied. S100A1/A4 proteins inhibit IFN-β-induced suppression of MCF-7 cells viability. The revealed direct influence of specific S100 proteins on IFN-β activity uncovers a novel regulatory role of particular S100 proteins, and opens up novel approaches to enhancement of therapeutic efficacy of IFN-β.     

Molecular Cloning and Characterization of the Calcineurin Subunit A from Plutella xylostella

Calcineurin (or PP2B) has been reported to be involved in an array of physiological process in insects, and the calcineurin subunit A (CNA) plays a central role in calcineurin activity. We cloned the CNA gene from Plutella xylostella (PxCNA). This gene contains an ORF of 1488 bp that encodes a 495 amino acid protein, showing 98%, and 80% identities to the CNA of Bombyx mori, and humans respectively. The full-length of PxCNA and its catalytic domain (CNA_1–341, defined as PxCNα) were both expressed in Escherichia coli. Purified recombinant PxCNA displayed no phosphatase activity, whereas recombinant PxCNα showed high phosphatase activity with a Km of 4.6 mM and a kcat of 0.66 S⁻¹ against pNPP. It could be activated at different degrees by Mn²⁺, Ni²⁺, Mg²⁺, and Ca²⁺. The optimum reaction pH was about 7.5 and the optimum reaction temperature was around 45 °C. An in vitro inhibition assay showed that okadaic acid (OA) and cantharidin (CTD) competitively inhibited recombinant PxCNα activity with the IC₅₀ values of 8.95 μM and 77.64 μM, respectively. However, unlike previous reports, pyrethroid insecticides were unable to inhibit recombinant PxCNα, indicating that the P. xylostella calcineurin appears not to be sensitive to class II pyrethroid insecticides.     

CK2 Down-Regulation Increases the Expression of Senescence-Associated Secretory Phenotype Factors through NF-κB Activation

Senescent cells secrete pro-inflammatory factors, and a hallmark feature of senescence is senescence-associated secretory phenotype (SASP). The aim of this study is to investigate the protein kinase CK2 (CK2) effects on SASP factors expression in cellular senescence and organism aging. Here CK2 down-regulation induced the expression of SASP factors, including interleukin (IL)-1β, IL-6, and matrix metalloproteinase (MMP) 3, through the activation of nuclear factor-κB (NF-κB) signaling in MCF-7 and HCT116 cells. CK2 down-regulation-mediated SIRT1 inactivation promoted the degradation of inhibitors of NF-κB (IκB) by activating the AKT-IκB kinase (IKK) axis and increased the acetylation of lysine 310 on RelA/p65, an important site for the activity of NF-κB. kin-10 (the ortholog of CK2β) knockdown increased zmp-1, -2, and -3 (the orthologs of MMP) expression in nematodes, but AKT inhibitor triciribine and SIRT activator resveratrol significantly abrogated the increased expression of these genes. Finally, antisense inhibitors of miR-186, miR-216b, miR-337-3p, and miR-760 suppressed CK2α down-regulation, activation of the AKT-IKK-NF-κB axis, RelA/p65 acetylation, and expression of SASP genes in cells treated with lipopolysaccharide. Therefore, this study indicated that CK2 down-regulation induces the expression of SASP factors through NF-κB activation, which is mediated by both activation of the SIRT1-AKT-IKK axis and RelA/p65 acetylation, suggesting that the mixture of the four miRNA inhibitors can be used as anti-inflammatory agents.     

Triterpenoids from the Roots of Rhaphiolepis indica var. tashiroi and Their Anti-Inflammatory Activity

Two new triterpenoids, 2α,3β-dihydroxyolean-11,13(18)-dien-19β,28-olide (1) and 3β,5β-dihydroxyglutinol (2), together with eight known compounds (3–10) were isolated from the roots of Rhaphiolepis indica var. tashiroi (Rosaceae). The structures of 1–10 were determined by spectroscopic techniques. Among these isolates, 2α,3β-dihydroxyolean-13(18)-en-28-oic acid (9) exhibited inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide production, with an IC₅₀ value of 16.50 μM.     

Prenylated Flavonoids from Cudrania tricuspidata Suppress Lipopolysaccharide-Induced Neuroinflammatory Activities in BV2 Microglial Cells

In Korea and China, Cudrania tricuspidata Bureau (Moraceae) is an important traditional medicinal plant used to treat lumbago, hemoptysis, and contusions. The C. tricuspidata methanol extract suppressed both production of NO and PGE₂ in BV2 microglial cells. Cudraflavanone D (1), isolated from this extract, remarkably suppressed the protein expression of inducible NO synthase and cyclooxygenase-2, and decreased the levels of NO and PGE₂ in BV2 microglial cells exposed to lipopolysaccharide. Cudraflavanone D (1) also decreased IL-6, TNF-α, IL-12, and IL-1β production, blocked nuclear translocation of NF-κB heterodimers (p50 and p65) by interrupting the degradation and phosphorylation of inhibitor of IκB-α, and inhibited NF-κB binding. In addition, cudraflavanone D (1) suppressed the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK pathways. This study indicated that cudraflavanone D (1) can be a potential drug candidate for the cure of neuroinflammation.     

Geranylgeraniol Inhibits Lipopolysaccharide-Induced Inflammation in Mouse-Derived MG6 Microglial Cells via NF-κB Signaling Modulation

Persistent inflammatory reactions in microglial cells are strongly associated with neurodegenerative pathogenesis. Additionally, geranylgeraniol (GGOH), a plant-derived isoprenoid, has been found to improve inflammatory conditions in several animal models. It has also been observed that its chemical structure is similar to that of the side chain of menaquinone-4, which is a vitamin K₂ sub-type that suppresses inflammation in mouse-derived microglial cells. In this study, we investigated whether GGOH has a similar anti-inflammatory effect in activated microglial cells. Particularly, mouse-derived MG6 cells pre-treated with GGOH were exposed to lipopolysaccharide (LPS). Thereafter, the mRNA levels of pro-inflammatory cytokines were determined via qRT-PCR, while protein expression levels, especially the expression of NF-κB signaling cascade-related proteins, were determined via Western blot analysis. The distribution of NF-κB p65 protein was also analyzed via fluorescence microscopy. Thus, it was observed that GGOH dose-dependently suppressed the LPS-induced increase in the mRNA levels of Il-1β, Tnf-α, Il-6, and Cox-2. Furthermore, GGOH inhibited the phosphorylation of TAK1, IKKα/β, and NF-κB p65 proteins as well as NF-κB nuclear translocation induced by LPS while maintaining IκBα expression. We showed that GGOH, similar to menaquinone-4, could alleviate LPS-induced microglial inflammation by targeting the NF-kB signaling pathway.     

Preparation and Characterization of a Chloroperoxidase-like Catalytic Antibody

The small molecule, meso-tetra(α,α,α,α-o-phenylacetamidophenyl) porphyrin (Mr1147.0) was used as complete antigen to elicit MAb through the immunization and cell fusion techniques. The MAb 1F2 obtained was demonstrated to be very pure by MALDI/TOFMS. The subtype of MAb 1F2 is IgG2a, which has a relative molecular weight of 156,678.8 Da.No significant change in the intensity of absorption peaks in UV and CD spectra was observed over a pH range between 6 and 12. The high stability of the abzyme and the tight binding between Fe porphyrin and antibody were also demonstrated. V_max, K_m, κcat, κcat/K_m for abzyme are 5.18 × 10⁻⁸ Ms⁻¹, 1.50 × 10⁻⁸ M, 0.518 s⁻¹, 3.45 × 10⁷ M⁻¹s⁻¹, respectively. The data obtained indicate that catalytic antibody has high catalytic activity. The chloroperoxidase activity of MAb 1F2-Fe porphyrin complex is stable from 10 °C to 60 °C.     

Structure,Dynamics, and Ligand Recognition of Human-Specific CHRFAM7A (Dupα7) Nicotinic Receptor Linked to Neuropsychiatric Disorders

Cholinergic α7 nicotinic receptors encoded by the CHRNA7 gene are ligand-gated ion channels directly related to memory and immunomodulation. Exons 5–7 in CHRNA7 can be duplicated and fused to exons A-E of FAR7a, resulting in a hybrid gene known as CHRFAM7A, unique to humans. Its product, denoted herein as Dupα7, is a truncated subunit where the N-terminal 146 residues of the ligand binding domain of the α7 receptor have been replaced by 27 residues from FAM7. Dupα7 negatively affects the functioning of α7 receptors associated with neurological disorders, including Alzheimer’s diseases and schizophrenia. However, the stoichiometry for the α7 nicotinic receptor containing dupα7 monomers remains unknown. In this work, we developed computational models of all possible combinations of wild-type α7 and dupα7 pentamers and evaluated their stability via atomistic molecular dynamics and coarse-grain simulations. We assessed the effect of dupα7 subunits on the Ca²⁺ conductance using free energy calculations. We showed that receptors comprising of four or more dupα7 subunits are not stable enough to constitute a functional ion channel. We also showed that models with dupα7/α7 interfaces are more stable and are less detrimental for the ion conductance in comparison to dupα7/dupα7 interfaces. Based on these models, we used protein–protein docking to evaluate how such interfaces would interact with an antagonist, α-bungarotoxin, and amyloid Aβ₄₂. Our findings show that the optimal stoichiometry of dupα7/α7 functional pentamers should be no more than three dupα7 monomers, in favour of a dupα7/α7 interface in comparison to a homodimer dupα7/dupα7 interface. We also showed that receptors bearing dupα7 subunits are less sensitive to Aβ₄₂ effects, which may shed light on the translational gap reported for strategies focused on nicotinic receptors in ‘Alzheimer’s disease research.     

A20 Overexpression Inhibits Lipopolysaccharide-Induced NF-κB Activation,TRAF6 and CD40 Expression in Rat Peritoneal Mesothelial Cells

Zinc finger protein A20 is a key negative regulator of inflammation. However, whether A20 may affect inflammation during peritoneal dialysis (PD)-associated peritonitis is still unclear. This study was aimed to investigate the effect of A20 overexpression on lipopolysaccharide (LPS)-induced inflammatory response in rat peritoneal mesothelial cells (RPMCs). Isolated and cultured RPMCs in vitro. Plasmid pGEM-T easy-A20 was transfected into RPMCs by Lipofectamine™2000. The protein expression of A20, phospho-IκBα, IκBα, TNF receptor-associated factor (TRAF) 6 and CD40 were analyzed by Western blot. The mRNA expression of TRAF6, CD40, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined by real time-PCR. NF-κB p65 DNA binding activity, IL-6 and TNF-α levels in cells culture supernatant were determined by ELISA. Our results revealed that RPMCs overexpression of A20 lead to significant decrease of LPS-induced IκBα phosphorylation and NF-κB DNA binding activity (all p < 0.01). In addition, A20 also attenuated the expression of TRAF6, CD40, IL-6 and TNF-α as well as levels of IL-6 and TNF-α in cells culture supernatant (all p < 0.05). However, A20 only partly inhibited CD40 expression. Our study indicated that A20 overexpression may depress the inflammatory response induced by LPS in cultured RPMCs through negatively regulated the relevant function of adaptors in LPS signaling pathway.     

Systematic Approach to Find the Global Minimum of Relaxation Dispersion Data for Protein-Induced B–Z Transition of DNA

Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion spectroscopy is commonly used for quantifying conformational changes of protein in μs-to-ms timescale transitions. To elucidate the dynamics and mechanism of protein binding, parameters implementing CPMG relaxation dispersion results must be appropriately determined. Building an analytical model for multi-state transitions is particularly complex. In this study, we developed a new global search algorithm that incorporates a random search approach combined with a field-dependent global parameterization method. The robust inter-dependence of the parameters carrying out the global search for individual residues (GSIR) or the global search for total residues (GSTR) provides information on the global minimum of the conformational transition process of the Zα domain of human ADAR1 (hZα_ADAR1)–DNA complex. The global search results indicated that a α-helical segment of hZα_ADAR1 provided the main contribution to the three-state conformational changes of a hZα_ADAR1—DNA complex with a slow B–Z exchange process. The two global exchange rate constants, k_ex and k_ZB, were found to be 844 and 9.8 s⁻¹, respectively, in agreement with two regimes of residue-dependent chemical shift differences—the “dominant oscillatory regime” and “semi-oscillatory regime”. We anticipate that our global search approach will lead to the development of quantification methods for conformational changes not only in Z-DNA binding protein (ZBP) binding interactions but also in various protein binding processes.     

Kansuinine A Ameliorates Atherosclerosis and Human Aortic Endothelial Cell Apoptosis by Inhibiting Reactive Oxygen Species Production and Suppressing IKKβ/IκBα/NF-κB Signaling

Reactive oxygen species (ROS)-induced vascular endothelial cell apoptosis is strongly associated with atherosclerosis progression. Herein, we aimed to examine whether Kansuinine A (KA), extracted from Euphorbia kansui L., prevents atherosclerosis development in a mouse model and inhibits cell apoptosis through oxidative stress reduction. Atherosclerosis development was analyzed in apolipoprotein E-deficient (ApoE^−/−) mice fed a high-fat diet (HFD) using Oil Red O staining and H&E staining. Human aortic endothelial cells (HAECs) were treated with KA, followed by hydrogen peroxide (H₂O₂), to investigate the KA-mediated inhibition of ROS-induced oxidative stress and cell apoptosis. Oil Red O staining and H&E staining showed that atherosclerotic lesion size was significantly smaller in the aortic arch of ApoE^−/− mice in the HFD+KA group than that in the aortic arch of those in the HFD group. Further, KA (0.1–1.0 μM) blocked the H₂O₂-induced death of HAECs and ROS generation. The H₂O₂-mediated upregulation of phosphorylated IKKβ, phosphorylated IκBα, and phosphorylated NF-κB was suppressed by KA. KA also reduced the Bax/Bcl-2 ratio and cleaved caspase-3 expression, preventing H₂O₂-induced vascular endothelial cell apoptosis. Our results indicate that KA may protect against ROS-induced endothelial cell apoptosis and has considerable clinical potential in the prevention of atherosclerosis and cardiovascular diseases.     

HIF-1α Dependent Upregulation of ZIP8, ZIP14, and TRPA1 Modify Intracellular Zn2+ Accumulation in Inflammatory Synoviocytes

Intracellular free zinc ([Zn²⁺]_i) is mobilized in neuronal and non-neuronal cells under physiological and/or pathophysiological conditions; therefore, [Zn²⁺]_i is a component of cellular signal transduction in biological systems. Although several transporters and ion channels that carry Zn²⁺ have been identified, proteins that are involved in Zn²⁺ supply into cells and their expression are poorly understood, particularly under inflammatory conditions. Here, we show that the expression of Zn²⁺ transporters ZIP8 and ZIP14 is increased via the activation of hypoxia-induced factor 1α (HIF-1α) in inflammation, leading to [Zn²⁺]_i accumulation, which intrinsically activates transient receptor potential ankyrin 1 (TRPA1) channel and elevates basal [Zn²⁺]_i. In human fibroblast-like synoviocytes (FLSs), treatment with inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), evoked TRPA1-dependent intrinsic Ca²⁺ oscillations. Assays with fluorescent Zn²⁺ indicators revealed that the basal [Zn²⁺]_i concentration was significantly higher in TRPA1-expressing HEK cells and inflammatory FLSs. Moreover, TRPA1 activation induced an elevation of [Zn²⁺]_i level in the presence of 1 μM Zn²⁺ in inflammatory FLSs. Among the 17 out of 24 known Zn²⁺ transporters, FLSs that were treated with TNF-α and IL-1α exhibited a higher expression of ZIP8 and ZIP14. Their expression levels were augmented by transfection with an active component of nuclear factor-κB P65 and HIF-1α expression vectors, and they could be abolished by pretreatment with the HIF-1α inhibitor echinomycin (Echi). The functional expression of ZIP8 and ZIP14 in HEK cells significantly increased the basal [Zn²⁺]_i level. Taken together, Zn²⁺ carrier proteins, TRPA1, ZIP8, and ZIP14, induced under HIF-1α mediated inflammation can synergistically change [Zn²⁺]_i in inflammatory FLSs.     

Downregulation of de Novo Fatty Acid Synthesis in Subcutaneous Adipose Tissue of Moderately Obese Women

The purpose of this work was to evaluate the expression of fatty acid metabolism-related genes in human adipose tissue from moderately obese women. We used qRT-PCR and Western Blot to analyze visceral (VAT) and subcutaneous (SAT) adipose tissue mRNA expression involved in de novo fatty acid synthesis (ACC1, FAS), fatty acid oxidation (PPARα, PPARδ) and inflammation (IL6, TNFα), in normal weight control women (BMI < 25 kg/m², n = 35) and moderately obese women (BMI 30–38 kg/m², n = 55). In SAT, ACC1, FAS and PPARα mRNA expression were significantly decreased in moderately obese women compared to controls. The downregulation reported in SAT was more pronounced when BMI increased. In VAT, lipogenic-related genes and PPARα were similar in both groups. Only PPARδ gene expression was significantly increased in moderately obese women. As far as inflammation is concerned, TNFα and IL6 were significantly increased in moderate obesity in both tissues. Our results indicate that there is a progressive downregulation in lipogenesis in SAT as BMI increases, which suggests that SAT decreases the synthesis of fatty acid de novo during the development of obesity, whereas in VAT lipogenesis remains active regardless of the degree of obesity.     

Potential Protection Effect of ER Homeostasis of N6-(2-Hydroxyethyl)adenosine Isolated from Cordyceps cicadae in Nonsteroidal Anti-Inflammatory Drug-Stimulated Human Proximal Tubular Cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) belong to a class of universally and commonly used anti-inflammatory analgesics worldwide. A diversity of drawbacks of NSAIDs have been reported including cellular oxidative stress, which in turn triggers the accumulation of unfolded proteins, enhancing endoplasmic reticulum stress, and finally resulting in renal cell damage. Cordyceps cicadae (CC) has been used as a traditional medicine for improving renal function via its anti-inflammatory effects. N⁶-(2-hydroxyethyl)adenosine (HEA), a physiologically active compound, has been reported from CC mycelia (CCM) with anti-inflammatory effects. We hypothesize that HEA could protect human proximal tubular cells (HK–2) from NSAID-mediated effects on differential gene expression at the mRNA and protein levels. To verify this, we first isolated HEA from CCM using Sephadex^® LH–20 column chromatography. The MTT assay revealed HEA to be nontoxic up to 100 µM toward HK–2 cells. The HK–2 cells were pretreated with HEA (10–20 µM) and then insulted with the NSAIDs diclofenac (DCF, 200 µM) and meloxicam (MXC, 400 µM) for 24 h. HEA (20 µM) effectively prevented ER stress by attenuating ROS production (p < 0.001) and gene expression of ATF–6, PERK, IRE1α, CDCFHOP, IL1β, and NFκB within 24 h. Moreover, HEA reversed the increase of GRP78 and CHOP protein expression levels induced by DCF and MXC, and restored the ER homeostasis. These results demonstrated that HEA treatments effectively protect against DCF- and MXC-induced ER stress damage in human proximal tubular cells through regulation of the GRP78/ATF6/PERK/IRE1α/CHOP pathway.     

New Insights into the Structure of Kappa/Beta-Carrageenan: A Novel Potential Inhibitor of HIV-1

New insights into the structure of the hybrid κ/β-carrageenan (κ/β-CRG) of the red alga Tichocarpus crinitus have been obtained. Carrageenan oligosaccharides were prepared through the chemical and enzymatic depolymerization of κ/β-CRG with κ-carrageenase and its the enzyme-resistant fraction. The composition and distribution of the repetition units of κ/β- CRG were investigated by using the negative ion tandem MALDI-TOFMS and ESIMS method, which made it possible to prove and characterize the hybrid structure of this polysaccharide. An analysis revealed the blockwise distribution of the long β-blocks along the polysaccharide chain, with the inclusion of κ/β, μ/ν-blocks and some ι-blocks. Furthermore, the desulfated κ/β-CRG was shown to contain of –G–D– repeating units up to 3.5 kDa. Previous studies have demonstrated that CRGs suppress the replication of several viruses. Here, we established that κ/β-CRG and its oligosaccharides significantly inhibit the transduction efficiency of replication-defective lentiviral particles pseudotyped with the envelope proteins of three different viruses. We found that the polysaccharide and its oligosaccharides strongly reduced the transduction efficiency of lentiviral particles pseudotyped with GP160—the envelope protein of the human immunodeficiency virus HIV-1—when added to T-lymphocyte Jurkat cells. The CRG oligosaccharides displayed significantly higher antiviral activity.