Hydrogen sulfide (H(2)S) is mainly known for its toxicity but has recently been shown to be produced endogenously in mammalian tissues and to be associated with physiological regulatory functions. To ...better understand the role of biomembranes in modulating its biological distribution and effects; we measured the partition coefficient of H(2)S in models of biological membranes. The partition coefficients were found to be 2.1±0.2, 1.9±0.5 and 2.0±0.6 in n-octanol, hexane and dilauroylphosphatidylcholine liposome membranes relative to water, respectively (25°C). This two-fold higher concentration of H(2)S in the membrane translates into a rapid membrane permeability, P(m) = 3 cm s(-1). We used a mathematical model in three dimensions to gain insight into the diffusion of total sulfide in tissues. This model shows that the sphere of action of sulfide produced by a single cell expands to involve more than 200 neighboring cells, and that the resistance imposed by lipid membranes has a significant effect on the diffusional spread of sulfide at pH 7.4, increasing local concentrations. These results support the role of hydrogen sulfide as a paracrine signaling molecule and reveal advantageous pharmacokinetic properties for its therapeutic applications.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Hydrogen sulfide (H2S) participates in prokaryotic metabolism and is associated with several physiological functions in mammals. H2S reacts with oxidized thiol derivatives (i.e. disulfides and ...sulfenic acids) and thereby forms persulfides, which are plausible transducers of the H2S-mediated signaling effects. The one-cysteine peroxiredoxin alkyl hydroperoxide reductase E from Mycobacterium tuberculosis (MtAhpE–SH) reacts fast with hydroperoxides, forming a stable sulfenic acid (MtAhpE–SOH), which we chose here as a model to study the interactions between H2S and peroxiredoxins (Prx). MtAhpE–SOH reacted with H2S, forming a persulfide (MtAhpE–SSH) detectable by mass spectrometry. The rate constant for this reaction was (1.4 ± 0.2) × 103m−1 s−1 (pH 7.4, 25 °C), six times higher than that reported for the reaction with the main low-molecular-weight thiol in M. tuberculosis, mycothiol. H2S was able to complete the catalytic cycle of MtAhpE and, according to kinetic considerations, it could represent an alternative substrate in M. tuberculosis. MtAhpE–SSH reacted 43 times faster than did MtAhpE–SH with the unspecific electrophile 4,4′-dithiodipyridine, a disulfide that exhibits no preferential reactivity with peroxidatic cysteines, but MtAhpE–SSH was less reactive toward specific Prx substrates such as hydrogen peroxide and peroxynitrite. According to molecular dynamics simulations, this loss of specific reactivity could be explained by alterations in the MtAhpE active site. MtAhpE–SSH could transfer its sulfane sulfur to a low-molecular-weight thiol, a process likely facilitated by the low pKa of the leaving thiol MtAhpE–SH, highlighting the possibility that Prx participates in transpersulfidation. The findings of our study contribute to the understanding of persulfide formation and reactivity.
The retropepsin (PR) of the Bovine leukemia virus (BLV) plays, as in other retroviruses, a crucial role in the transition from the non-infective viral particle to the infective virion by processing ...the polyprotein Gag. PR is expressed as an immature precursor associated with Gag, after an occasional -1 ribosomal frameshifting event. Self-hydrolysis of PR at specific N- and C-terminal sites releases the monomer that dimerizes giving rise to the active protease. We designed a strategy to express BLV PR in E. coli as a fusion protein with maltose binding protein, with a six-histidine tag at its N-terminal end, and bearing a tobacco etch virus protease hydrolysis site. This allowed us to obtain soluble and mature recombinant PR in relatively good yields, with exactly the same amino acid composition as the native protein. As PR presents relative promiscuity for the hydrolysis sites we designed four fluorogenic peptide substrates based on Förster resonance energy transfer (FRET) in order to characterize the activity of the recombinant enzyme. These substrates opened the way to perform kinetic studies, allowing us to characterize the dimer-monomer equilibrium. Furthermore, we obtained kinetic evidence for the existence of a conformational change that enables the interaction with the substrate. These results constitute a starting point for the elucidation of the kinetic properties of BLV-PR, and may be relevant not only to improve the chemical warfare against this virus but also to better understand other viral PRs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human embryonic stem cells (hESCs) are an attractive resource for new therapeutic approaches that involve tissue regeneration. hESCs have exhibited low immunogenicity due to low levels of Mayor ...Histocompatibility Complex (MHC) class-I and absence of MHC class-II expression. Nevertheless, the mechanisms regulating MHC expression in hESCs had not been explored.
We analyzed the expression levels of classical and non-classical MHC class-I, MHC class-II molecules, antigen-processing machinery (APM) components and NKG2D ligands (NKG2D-L) in hESCs, induced pluripotent stem cells (iPSCs) and NTera2 (NT2) teratocarcinoma cell line. Epigenetic mechanisms involved in the regulation of these genes were investigated by bisulfite sequencing and chromatin immunoprecipitation (ChIP) assays. We showed that low levels of MHC class-I molecules were associated with absent or reduced expression of the transporter associated with antigen processing 1 (TAP-1) and tapasin (TPN) components in hESCs and iPSCs, which are involved in the transport and load of peptides. Furthermore, lack of beta2-microglobulin (beta2m) light chain in these cells limited the expression of MHC class I trimeric molecule on the cell surface. NKG2D ligands (MICA, MICB) were observed in all pluripotent stem cells lines. Epigenetic analysis showed that H3K9me3 repressed the TPN gene in undifferentiated cells whilst HLA-B and beta2m acquired the H3K4me3 modification during the differentiation to embryoid bodies (EBs). Absence of HLA-DR and HLA-G expression was regulated by DNA methylation.
Our data provide fundamental evidence for the epigenetic control of MHC in hESCs and iPSCs. Reduced MHC class I and class II expression in hESCs and iPSCs can limit their recognition by the immune response against these cells. The knowledge of these mechanisms will further allow the development of strategies to induce tolerance and improve stem cell allograft acceptance.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Natural Killer Group 2 member D (NKG2D) activating receptor, present on the surface of various immune cells, plays an important role in activating the anticancer immune response by their interaction ...with stress-inducible NKG2D ligands (NKG2DL) on transformed cells. However, cancer cells have developed numerous mechanisms to evade the immune system via the downregulation of NKG2DL from the cell surface, including the release of NKG2DL from the cell surface in a soluble form. Here, we review the mechanisms involved in the production of soluble NKG2DL (sNKG2DL) and the potential therapeutic strategies aiming to block the release of these immunosuppressive ligands. Therapeutically enabling the NKG2D-NKG2DL interaction would promote immunorecognition of malignant cells, thus abrogating disease progression.
B7-H6, a ligand for the NK activating receptor NKp30, has been identified as a biomarker of poor prognosis in several solid cancers. However, little is known about the role of B7-H6 and the ...mechanisms that control its expression in acute myeloid leukemia (AML). Epigenome modulation, including epigenomic reader dysregulation, is one of the hallmarks of AML. Bromodomain-containing protein 4 (BRD4), the best-known member of the BET family of epigenetic readers, is overexpressed in AML cells and regulates the transcription of genes involved in the pathogenesis of AML, as MYC oncogene. Here, we analyze the role of BRD4 in regulating B7-H6 in AML cells. Results demonstrated that the specific inhibition of BRD4 drastically reduces the expression of B7-H6 in AML cells. Histone acetylation mediated by CBP30/P300 facilitates the binding of BRD4 to the B7-H6 promoter, which recruits the P-TEFb elongation factor that phosphorylates RNA polymerase II, thereby activating B7-H6 transcription. BRD4 also co-bounded with JMJD6 at the distal enhancer of the B7-H6 gene. Metabolic modulation with metformin modifies the acetylation pattern in the B7-H6 promoter, impairing BRD4 binding, thereby inhibiting B7-H6 expression. B7-H6 knockdown induces the apoptosis in HEL-R cell line. Moreover, a high level of B7-H6 expression in AML patients is related to increased BRD4 levels, myelodysplastic-derived AML, and del5q, the two latter being associated with poor prognosis. Our data show that BRD4 is a positive regulator of the pro-tumorigenic molecule B7-H6 and that the blockage of the B7-H6 is a potential therapeutic target for the treatment of AML.
Primary/secondary hyperoxalurias involve nephrocalcinosis-related chronic kidney disease (CKD) leading to end-stage kidney disease. Mechanistically, intrarenal calcium oxalate crystal deposition is ...thought to elicit inflammation, tubular injury and atrophy, involving the NLRP3 inflammasome. Here, we found that mice deficient in NLRP3 and ASC adaptor protein failed to develop nephrocalcinosis, compromising conclusions on nephrocalcinosis-related CKD. In contrast, hyperoxaluric wild-type mice developed profound nephrocalcinosis. NLRP3 inhibition using the β-hydroxybutyrate precursor 1,3-butanediol protected such mice from nephrocalcinosis-related CKD. Interestingly, the IL-1 inhibitor anakinra had no such effect, suggesting IL-1-independent functions of NLRP3. NLRP3 inhibition using 1,3-butanediol treatment induced a shift of infiltrating renal macrophages from pro-inflammatory (CD45+F4/80+CD11b+CX3CR1+CD206-) and pro-fibrotic (CD45+F4/80+CD11b+CX3CR1+CD206+TGFβ+) to an anti-inflammatory (CD45+F4/80+CD11b+CD206+TGFβ-) phenotype, and prevented renal fibrosis. Finally, in vitro studies with primary murine fibroblasts confirmed the non-redundant role of NLRP3 in the TGF-β signaling pathway for fibroblast activation and proliferation independent of the NLRP3 inflammasome complex formation. Thus, nephrocalcinosis-related CKD involves NLRP3 but not necessarily via intrarenal IL-1 release but rather via other biological functions including TGFR signaling and macrophage polarization. Hence, NLRP3 may be a promising therapeutic target in hyperoxaluria and nephrocalcinosis.
Global mechanisms defining the gene expression programs specific for hematopoiesis are still not fully understood. Here, we show that promoter DNA demethylation is associated with the activation of ...hematopoietic-specific genes. Using genome-wide promoter methylation arrays, we identified 694 hematopoietic-specific genes repressed by promoter DNA methylation in human embryonic stem cells and whose loss of methylation in hematopoietic can be associated with gene expression. The association between promoter methylation and gene expression was studied for many hematopoietic-specific genes including CD45, CD34, CD28, CD19, the T cell receptor (TCR), the MHC class II gene HLA-DR, perforin 1 and the phosphoinositide 3-kinase (PI3K) and results indicated that DNA demethylation was not always sufficient for gene activation. Promoter demethylation occurred either early during embryonic development or later on during hematopoietic differentiation. Analysis of the genome-wide promoter methylation status of induced pluripotent stem cells (iPSCs) generated from somatic CD34+ HSPCs and differentiated derivatives from CD34+ HSPCs confirmed the role of DNA methylation in regulating the expression of genes of the hemato-immune system, and indicated that promoter methylation of these genes may be associated to stemness. Together, these data suggest that promoter DNA demethylation might play a role in the tissue/cell-specific genome-wide gene regulation within the hematopoietic compartment.
Acute myeloid leukemia (AML) is a heterogeneous disease whose therapies currently show elevated toxicity and a high rate of relapse. Recently, the burgeoning of new anti-tumor therapeutic strategies ...aimed at enhancing the immune response has pushed natural killer cells (NKs) into the spotlight. These cells are powerful warriors that can bring about the lysis of tumor cells through their cytotoxic ability. However, tumor cells have developed strategies to evade recognition mediated by NKs. Here, we review the mechanisms triggered by AML cells and discuss the emerging immunotherapeutic strategies that potentiate the anti-tumor functions of NKs.
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