Isogarcinol (YDIS), a natural compound extracted from Garcinia mangostana L., has a significant immunosuppressive effect on systemic lupus erythematosus and rheumatoid arthritis. This paper reports that it reduced imiquimod-induced psoriasis-like skin lesions in mice. It strongly attenuated the aberrant proliferation and differentiation of keratinocytes. Moreover, the expression of genes involving the interleukin-23 (IL-23)/T-helper 17 (Th17) axis was significantly inhibited in the dorsal skin of the YDIS-treated mice, as was that of the other pro-inflammatory factors TNF-α, IL-2, and even interferon (IFN)-γ. Furthermore, YDIS prevented the abnormal distribution of T cell types and suppressed the differentiation of CD4+ T cells into Th17 cells in the spleens of mice exposed to imiquimod. Interestingly, it elevated numbers of regulatory T cells (Tregs) in the spleen and boosted IL-10 expression in the skin. In agreement with the above, YDIS increased serum IL-10 and reduced serum IL-17. It also caused less damage to the liver and, especially, kidneys of mice than cyclosporine A (CsA). In vitro, YDIS caused more death of HaCaT keratinocytes than CsA. It also strongly inhibited inflammatory factor expression in lipopolysaccharide (LPS)-stimulated HaCaT cells. These findings suggest that YDIS is a promising immunosuppressive agent for treating psoriasis.Keywords: cyclosporine A; HaCaT; imiquimod-induced; isogarcinol; psoriasis; regulatory T cell; T-helper 17 cell;

•Quercitrin (YDHH) can attenuate IMQ-induced psoriasis-like skin lesions in mice.•Transcripts of cytokines on the IL-23/Th17 axis were decreased after YDHH treatment.•YDHH inhibits Th17 cell response via the JAK/STAT signalling pathway.•Compared with CsA, quercitrin causes less toxicity to the liver and kidneys.Quercitrin (YDHH) is a natural dietary flavonoid extracted from Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn.). Psoriasis is a chronic immune-mediated dermatosis. In this study, we report that YDHH had no obvious cytotoxicity and inhibited the proliferation of LPS-stimulated HaCaT keratinocytes. Furthermore, oral administration of YDHH alleviated imiquimod-induced psoriasis-like dermatitis in mice, significantly attenuating the excessive proliferation and abnormal differentiation of keratinocytes, and reducing inflammatory cell infiltrates. YDHH treatment lowered the expression of cytokines related to psoriasis, especially those on the IL-23/Th17 axis. Furthermore, it dramatically inhibited the Th17 cell response regulated by the JAK/STAT signalling pathway. In addition, it was less toxic to the liver and kidneys of mice than the classical immunosuppressant cyclosporine A. Taken together, these findings indicate that YDHH could be useful as a functional food supplement for protection against psoriasis.

Isogarcinol is a new natural immunosuppressant that was extracted from Garcinia mangostana L. in our laboratory. Knowledge of its effects on treatable diseases and its mechanism of action is still very limited. In this study, we explored the therapeutic effect of isogarcinol in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). Treatment with oral 100 mg/kg isogarcinol markedly ameliorated clinical scores, alleviated inflammation and demyelination of the spinal cord, and reduced intracranial lesions in EAE mice. The percentages of Th cells and macrophages were also strongly reduced. Isogarcinol appeared to act by inhibiting T helper (Th) 1 and Th17 cell differentiation via the janus kinase/signal transducers and activators of transcription pathway and by impairing macrophage function. Our data suggest that isogarcinol has the potential to be an effective therapeutic agent of low toxicity for treating MS and other autoimmune diseases.Keywords: cytokines; experimental autoimmune encephalomyelitis; Garcinia mangostana L.; isogarcinol; Jak/STAT pathway; multiple sclerosis;

The aim of this study was to establish a quality-control method for calcineurin subunit B (CNB) biological activity determinations. CNB enhances the p-nitrophenylphosphate (pNPP) dephosphorylating activity of calcineurin subunit A Δ316 mutant (CNAΔ316). A series of CNB concentrations were fitted to a four-parameter equation to calculate the corresponding pNPP maximum dephosphorylation rates. Values were calculated based on biological activity references using a parallel line method. The method was then validated for accuracy, precision, linearity, linear range, sensitivity, specificity, and robustness. The recovery results were greater than 98%. Intra-plate precision was 6.7%, with inter-plate precision of 10.8%. The coefficient of determination was greater than 0.98. The linear range was 0.05–50 μg mL-1, with sensitivity of 50 μg mL-1. Tested cytokines did not induce CNAΔ316 dephosphorylation of pNPP. The chosen CNAΔ316 concentration range did not affect activity determinations.

The calcineurin B subunit (CnB) is the regulatory subunit of Cn, a Ca2+/calmodulin-dependent serine/threonine protein phosphatase. In this study, we demonstrate that extracellular CnB was effectively internalized through a CD14-independent Toll-like receptor 4 (TLR4) pathway, which led to the phosphorylation of nuclear factor (NF)-kappa-B inhibitor alpha (IκB-α) and upregulation of pro-inflammatory cytokines in human monocytes. CnB-induced IκB-α phosphorylation is completely dependent on TNF receptor-associated factor 3 (TRAF3) but not TRAF6, which is indispensable for IκB-α phosphorylation in response to lipopolysaccharide. The loss-of-function CnB mutants were able to induce IκB-α phosphorylation, further indicating that this novel role of CnB is completely independent of the phosphatase function of Cn. Taken together, these findings demonstrate that CnB is a novel host-derived immunostimulatory factor, having a role as an agonist in monocytes, and specificity in TLR4 signaling through TRAF3 and TRAF6, in response to various agonists.

Macrophages are involved in tumor growth and progression. They infiltrate into tumors and cause inflammation, which creates a microenvironment favoring tumor growth and metastasis. However, certain stimuli may induce macrophages to act as tumor terminators. Here we report that the calcineurin B subunit (CnB) synergizes with IFN-γ to make macrophages highly cytotoxic to cancer cells. Furthermore, CnB and IFN-γ act synergistically to polarize mouse tumor-associated macrophages, as well as human monocyte-derived macrophages to an M1-like phenotype. This synergy is mediated by the crosstalk between CnB-engaged integrin αM-p38 MAPK signaling and IFN-γ-initiated p38/PKC-δ/Jak2 signaling. Interestingly, the signal transducer and activator of transcription 1 (STAT1) is a key factor that orchestrates the synergy of CnB and IFN-γ, and the phosphorylation status at Ser727 and Tyr701 of STAT1 is directly regulated by CnB and IFN-γ.

Isogarcinol is a new immunosuppressant that we extracted from Garcinia mangostana L. In the present study, we elucidate its beneficial effect in chronic graft-versus-host disease (cGVHD) in mice -- a model for systemic lupus erythematosus (SLE) in human. The oral administration of 60 mg/kg isogarcinol significantly reduced proteinuria, corrected the abnormal serum biochemical indicator, and decreased the amount of serum antibodies and lowered the renal histopathology score. In addition, isogarcinol alleviated the abnormal activation of CD4 T cells and decreased the expression of inflammatory genes and cytokines in the kidneys and peritoneal macrophages. The mechanism of action of isogarcinol is associated with downregulation of CD4 T cells and inflammatory effects. Therefore, we believe that isogarcinol may be a potential therapeutic drug candidate for future treatment of SLE.

Isogarcinol is a natural compound that we extracted from Garcinia mangostana L., and we were the first to report that it is a new immunosuppressant. In the present study, we investigated the immune regulation and anti-inflammatory effects of isogarcinol on collagen-induced arthritis (CIA) and explored its potential mechanism in the treatment of rheumatoid arthritis. The oral administration of isogarcinol significantly reduced clinical scores, alleviated cartilage and bone erosion, and reduced the levels of serum inflammatory cytokines in CIA mice. Isogarcinol inhibited xylene-induced mouse ear edema in vivo. In vitro, isogarcinol decreased iNOS and COX-2 mRNA expression and NO content by inhibiting NF-κB expression. Furthermore, isogarcinol decreased the activity of NFAT and inhibited IL-2 expression. The mechanism of action of isogarcinol is associated with down-regulation of both autoimmune and inflammatory reactions.

Calcineurin B subunit (CnB) is the regulatory subunit of calcineurin (Cn), a Ca2+/calmodulin-dependent serine/threonine protein phosphatase. It has been reported that mice deleting the CnB gene lose nearly all Cn activity and show poor tolerance to cardiac stress; CnB gene expression is downregulated in the hearts of rats that have suffered ischemia/reperfusion (I/R) injury. Therefore, we wonder whether injection of exogenous CnB protein can prevent the rats from suffering I/R injury. In cardiomyocytes, fluorogenic labeling shows that exogenous CnB quickly enters the cell. Pretreatment of cardiomyocytes with CnB reduces apoptosis in response to hypoxia/reoxygenation injury (an in vitro model mimicking ischemia/reperfusion injury), and CsA reverses this effect by inhibiting Cn activity. Furthermore, CnB upregulates Bcl-2 and Bcl-XL expression in the process of hypoxia/reoxygenation injury, which may contribute to protecting cardiomyocytes against apoptosis. In vivo experiments shows that pretreatment with CnB improves cardiac contractile function and reduces the frequency of arrhythmias induced by global I/R injury. These findings reveal a novel function for CnB protein in cardiac stress response and suggest a possible application of CnB in coronary disease therapy.

PP1, PP2A and PP2B all belong to PPP family of serine/threonine protein phosphatases. Their primary structures are highly conserved, particularly in the catalytic domain. In order to obtain correlative information about this conserved region, we constructed N-, C-deletion and N/C double-deletion mutants. We found that the N- and Csingle-deletion mutants exhibited higher enzymatic activities, while specific activity of N/C double-deletion mutant PP1 (9-306) did not notably change. The results of kinetics analysis showed that kcat and kcat/Km increased about 16-fold in the single-deletion mutants; while the two parameters of the double-deletion were lower than the single-deletions. We further explored stability of all mutants in existing denaturant guanidine hydrochloride (GdnHCl). It was noticeable that stability of PP1-(9-306) in all mutants was the highest. We speculated that PP1-(9-306) maybe retains a compact spherical structure, thus accordingly affected molecular catalysis. On the other hand the structures of single-deletion mutants were relatively relaxed, which were able to bind substrate easily, so activities of single-deletion mutants were higher than that of double-deletion mutant. We therefore deduced that PP1-(9-306) may be close to core region of PP1 molecule. In order to further solidify this idea, we used fluorescence spectra method to explore changes of space conformation. We found that emission peaks of all single-deletions were blue shifted in different degree in the absence of denaturant, while emission peak of N/C double-deletion mutant did not change obviously compared with that of the wild-type PP1. Conformation change of N/C double-deletion mutant was significantly less than those of single-deletion mutants in different GdnHCl concentration.

A novel protease, from the edible fungus Cordyceps sinensis, was purified and characterised. Its cleavage site motifs were determined by oriented peptide library mixtures and validated by synthetic peptides and natural proteins.The protease was purified to homogeneity using anion-exchange chromatography, sieve chromatography, native PAGE and reversion phase chromatography. Its molecular weight, estimated by SDS–PAGE, was approximately 43 kDa. The results of MS-MS, MALDI-TOF MS and de novo sequencing demonstrated that it was a completely new protease.We used oriented peptide library mixtures to determine cleavage site motifs. Cleavage requires lysine at P1 and proline or lysine at P3′. P2 and P1′ also show some preference. A series of synthetic peptides and natural proteins were used to validate the substrate specificity. The protease has special substrate preferences different from other proteases. It also has excellent biochemical properties, which make it able to withstand harsh conditions and suitable for industrialisation and commercialisation.Research highlights► A novel protease from Cordyceps sinensis was purified and characterised. ► Its cleavage site motifs were determined by oriented peptide library mixtures. ► The novel protease has completely new cleavage pattern -/-/-/K+-/-/PK/-.

Calcineurin is composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). CNA contains the catalytic domain and three regulatory domains: a CNB-binding domain (BBH), a C-terminal calmodulin-binding domain (CBD), and an autoinhibitory domain (AID). We constructed a series of mutants of CNA to explore the regulatory role of its C-terminal regulatory domain and CaM. We demonstrated a more precise mechanism of CNA regulation by C-terminal residues 389–511 in the presence of CNB. First, we showed that residues 389–413, which were identified in previous work as constituting a CaM binding domain (CBD), also have an autoinhibiting function. We also found that residues 389–413 were not sufficient for CaM binding and that the CBD comprises at least residues 389−456. In conclusion, two distinct segments of the C-terminal regulatory region (389–511) of CNA inhibit enzyme activity: residues 389–413 interact with the CNB binding helix (BBH), and residues 457–482 with the active center of CNA.

Solution 1H NMR spectroscopy has been carried out to investigate the molecular and electronic structures of the active site in H64Q/V68F double mutant mouse neuroglobin in the cyanomet form. Two heme orientations resulting from a 180° rotation about the α–γ-meso axis were observed with a population ratio about 1:1, and the clearly distinguished B isomer was used to perform the study. Based on the analysis of the dipolar shifts and paramagnetic relaxation constants, the distal Gln64(E7) side chain is obtained to adopt an orientation that may produce hydrogen bond between the NεH1 and the Fe-bound cyanide. The side chain of Phe68(E11) is oriented out of the heme pocket just like that in triple mutant of cyanide complex of sperm whale myoglobin. A 15° rotation of the imidazole ring in axial His96 is observed, which is close to the ϕ angle determined from the crystal structure of NgbCO. The quantitative determinations of the orientation and anisotropies of the paramagnetic susceptibility tensor reveal that cyanide is tilted by 8° from the heme normal which allows for contact to the Gln64(E7) NεH1. The E7 and E11 residues appear to control the direction and the extent of tilt of the bound ligand. Furthermore, the tilt of the ligand has no obvious influence on the heme heterogeneity of cyanide ligation for isomer A/B of the wild type and mutant protein, indicating that factors other than steric effects, such as polarity of heme pocket, impacts on ligand binding affinity.

In this study, an electrochemical method to assay calcineurin activity is proposed. Although the enzyme could not exhibit electron transfer reactivity and the catalytic reaction of the substrate could not give any electrochemical wave, p-nitrophenol as the catalytic reaction production could be oxidized at the calcineurin/Triton X-100 film modified electrode to exhibit useful wave that might be employed to assay the enzyme activity. The effect of Ni2+ and Zn2+ on calcineurin was also investigated. Whereas Ni2+ was confirmed to be able to enhance the enzymatic activity, Zn2+ was found to be an inhibitor to calcineurin.

PP1, PP2A and PP2B, belonging to the PPP family of Ser/Thr protein phosphatases, participate in regulating many important physiological processes, such as cell cycle control, regulation of cell growth and division regulation, etc. The sequence homology between them is relatively high, and tertiary structure is conserved. Because of the complexity of the structure of PP2A and the diversity of its regulatory subunits, its structure is less well known than those of PP1 and PP2B. The PP2A holoenzyme consists of a heterodimeric core enzyme, comprising a scaffolding subunit and a catalytic subunit, as well as a variable regulatory subunit. In this study, the subunit compositions, similarities and differences between the Ser/Thr protein phsphatases structures are summarized.

Protective immunity involves a dynamic balance between humoral and cellular immune responses. In the present work we demonstrated that recombinant human calcineurin subunit B (rhCnB) stimulated the expression of the surface molecules CD83, CD80, CD86, CD40, and HLA-DR. It also promoted secretion of inflammatory cytokines IL-6, TNF-α, and IL-1β by human PBMC-derived dendritic cells. In in vivo experiments, splenocytes from BALB/c mice immunized with pneumolysin plus rhCnB contained a higher percentage of CD3+CD4+ T lymphocytes, produced more antigen-specific splenocyte proliferation activity, and had higher anti-pneumolysin immunoglobulin G (IgG) titers. Transcript levels of cytokines such as IL-4, IL-10, and IFN-γ in the splenocytes were also upregulated when in vitro stimulated with pneumolysin. Thus, rhCnB promoted a mixed Th1/Th2 type immune response when given together with the specific antigen PN. RhCnB could have potential as a prophylactic vaccine adjuvant.Highlights• We used human PBMC-derived and mice marrow-derived dendritic cells as models. • We demonstrated that recombinant human calcineurin subunit B (CnB) induced phenotypic maturation of human PBMC-DCs and increased the secretion of cytokines. • In vivo experiments, we used murine models to examine the adjuvant effect of CnB on recombinant pneumolysin antigen (PN) specific immune responses. • It effectively promoted mixed Th1/Th2 type immune response.

A new convenient approach has been designed to produce polyclonal antibodies (PcAb). The approach is based on the principle of the immunoglobulin (Ig) class switch in the immune response. We produced six different antibodies (Ab) against calcineurin A subunit (CNA). CNA, His-tagged calcineurin A subunit (His-CNA), single chain calcineurin (CNB-CNA) and single chain calcineurin–calmodulin complex (CaM-CNB-CNA) were expressed in Escherichia coli (E. coli) BL21 strain, and they were used to immunize male BALB/c mice. These Ab were examined by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. The results clearly demonstrated that the specificity of the Ab produced by different protein samples was much higher than that of the Ab produced by a single sample. We used CNA, CaM-CNB-CNA and His-CNA to immunize mice in turn and obtained monospecific PcAb against CNA fortunately by our new approach. Remarkably, our approach not only offered a simple and general alternative to other methods for producing PcAb described previously, but also disclosed a novel process of immunization that could be used to produce monoclonal antibodies (mAb).