Vadim V. Sumbayev

S-nitrosation of Cys-800 of HIF-1α protein activates its interaction with p300 and stimulates its transcriptional activity

Hypoxia inducible factor 1 (HIF‐1) is a heterodimeric transcriptional complex that plays pivotal role in the regulation of cellular utilization of oxygen as well as glucose and is an essential regulator of angiogenesis in solid tumor and ischemic disorders. Recently HIF‐1α, a subunit of HIF‐1 complex, was characterized as a potential target for S‐nitrosation, providing no information about the impact of this posttranslational modification on the protein transactivation. Cys‐800 of HIF‐1α protein has reactive SH‐group, which is critical for the recruitment of p300 co‐activator that is necessary for transcriptional activity of HIF‐1 complex. Here we report that S‐nitrosation of Cys‐800 activates HIF‐1α–p300 interaction and stimulates protein transactivation. We have found that S‐nitrosation of the HIF‐1α C‐terminal domain by nitric oxide derived from donors and nitric oxide synthase increases protein transcriptional activity. The increase of HIF‐1 transcriptional activity was not observed in the case of Cys‐800 substitution to Ala, though other protein thiol groups were nitrosated. Experiments with GST pull‐down assay suggest that S‐nitrosation of Cys‐800 stimulates the recruitment of p300 co‐activator protein to the HIF‐1α C‐terminal domain.

S-nitrosylation of thioredoxin mediates activation of apoptosis signal-regulating kinase 1.

Apoptosis signal-regulating kinase 1 (ASK1) was recently discovered as a typical member of the mitogen-activated protein (MAP) kinase kinase kinase family, which induces apoptosis by activation of c-Jun-N-terminal kinase/p38 MAP kinase pathways. In normal cells ASK1 is directly inhibited by thioredoxin (Trx), a 12-kDa protein ubiquitously expressed in all living cells, which has a variety of biological functions related to cell proliferation and apoptosis. Here we found that purified Trx is sensitive to S-nitrosylation. Stimulation of HEK-293 cells with S-nitrosoglutathione (GSNO) for 2, 4, 8, and 16h also caused Trx S-nitrosylation, which showed straight correlation with ASK1 activation based on Western blot detection of the enzyme, immunoprecipitation assay, and measurement of its catalytic activity. These results suggest that S-nitrosylation of Trx induces ASK1 activation. Treatment of cells with N-acetyl-cysteine for 2h after 8h of pretreatment with GSNO caused an increase in glutathione and nullified ASK1 activation.

Role of MAP kinase-dependent apoptotic pathway in innate immune responses and viral infection.

Mitogen‐activated protein (MAP) kinase cascades are multifunctional signalling networks that influence cell growth, differentiation, apoptosis and cellular responses to stress. Apoptosis signal‐regulating kinase 1 (ASK1) is a MAP kinase kinase kinase that triggers apoptogenic kinase cascade leading to the phosphorylation/activation of c‐Jun N‐terminal kinases (JNK) and p38‐MAP kinase, which are responsible to induce apoptotic cell death. This pathway plays a pivotal role in the transduction of signals from different apoptotic stimuli. Recently, it has become evident that ASK1 and its downstream pathway are employed in the transduction of signals from Toll‐like receptors (TLR) – multistep processes that interfere with different intracellular signalling pathways. TLR are the key proteins that allow mammals to detect pathogens and mediate innate immune responses. In addition, ASK1 and its downstream pathway play a target role in the regulation of apoptosis in some cases of viral infection – AIDS, influenza, hepatitis C and others. In the present review, we summarize current knowledge about the role of ASK1 and its downstream pathway in innate immune responses and viral infection.

LPS-induced Toll-like receptor 4 signalling triggers cross-talk of apoptosis signal-regulating kinase 1 (ASK1) and HIF-1α protein

Toll‐like receptor 4 (TLR4) is required for recognition of lipopolysaccharide (LPS) of Gram‐negative bacteria and induction of the innate immune response to them. Nevertheless, the involvement of some crucial pathways in TLR4 signalling is poorly understood. Here, we report that LPS‐induced TLR4 signalling triggers cross talk of HIF‐1α and ASK1 in THP‐1 human myeloid monocytic leukaemia cells. Both pathways are activated via redox‐dependent mechanism associated with tyrosine kinase/phospholipase C‐1γ‐mediated activation of protein kinase C α/β, which are known to activate NADPH oxidase and the production of reactive oxygen species that activate both HIF‐1α and ASK1. ASK1 contributes to the stabilisation of HIF‐1α, most likely via activation of p38 MAP kinase.

Gold Nanoparticles Downregulate Interleukin‐1β‐Induced Pro‐Inflammatory Responses

Interleukin 1 beta (IL-1β)-dependent inflammatory disorders, such as rheumatoid arthritis and psoriasis, pose a serious medical burden worldwide, where patients face a lifetime of illness and treatment. Organogold compounds have been used since the 1930s to treat rheumatic and other IL-1β-dependent diseases and, though their mechanisms of action are still unclear, there is evidence that gold interferes with the transmission of inflammatory signalling. Here we show for the first time that citrate-stabilized gold nanoparticles, in a size dependent manner, specifically downregulate cellular responses induced by IL-1β both in vitro and in vivo. Our results indicate that the anti-inflammatory activity of gold nanoparticles is associated with an extracellular interaction with IL-1β, thus opening potentially novel options for further therapeutic applications.

The involvement of sphingosine kinase 1 in LPS-induced Toll-like receptor 4-mediated accumulation of HIF-1α protein, activation of ASK1 and production of the pro-inflammatory cytokine IL-6.

Toll‐like receptors (TLRs) lie in the core of resistance to infectious diseases allowing host immune cells to specifically detect pathogens by recognising their specific molecular patterns. Cell membrane‐associated TLR4 (recognises lipopolysaccharide (LPS) of Gram‐negative bacteria) and endosomal TLR7/8 (recognise viral single‐stranded RNA) are known to activate hypoxia inducible factor‐1α (HIF‐1α) protein (necessary for cellular adaptation to the inflammatory stress) via redox‐dependent mechanism. TLR4 triggers the cross talk between HIF‐1α and apoptosis signal‐regulating kinase 1 (ASK1), whereas TLR7/8 activates HIF‐1α in the ASK1‐independent manner. Here, we report that in THP‐1 and RAW264.7 macrophages, ligand‐induced activation of the TLR4 but not TLR7/8 induces activation and transcriptional upregulation of sphingosine kinase 1 (SphK1) in extracellular signal‐regulating kinase and phospholipase C‐1γ/PI3 kinase‐dependent manner. TLR4‐mediated SphK1 activation was found to be critical for the redox‐dependent activation of HIF‐1α and ASK1, as well as for the prevention of LPS‐induced activation of caspase 3 and the expression of pro‐inflammatory cytokine interleukin‐6.

HIF-1α protein is an essential factor for protection of myeloid cells against LPS-induced depletion of ATP and apoptosis that supports Toll-like receptor 4-mediated production of IL-6

Sepsis is the leading cause of death in intensive care units, which reflects detrimental host response to infection where lipopolysaccharide (LPS) shared by Gram-negative bacteria acts as a potent activator of immune cells via Toll-like receptor 4 (TLR4). Recently it was found that TLR4 downstream signalling leads to the accumulation of hypoxia-inducible factor 1 alpha (HIF-1alpha), which is important for TLR4-dependent expression of pro-inflammatory cytokines, however, basic biochemical mechanisms of involvement of this protein in TLR4 downstream signalling remains unclear. Here we found that knockdown of the expression of HIF-1alpha protein by siRNA led to the depletion of ATP, which corresponded to the constant increase in the activity of apoptosis signal-regulating kinase 1 (ASK1) and therefore apoptosis as estimated based on the increase in the activity of caspase 3. On the other hand, LPS-dependent production of IL-6 was attenuated. Treatment of HIF-1alpha knockdown cells with extracellular ATP in combination with LPS preserved the IL-6 expression but not the activity of ASK1 on the level observed in LPS-stimulated control cells. We therefore suggested that HIF-1alpha protein supports LPS-dependent expression of IL-6 by preventing depletion of ATP. On the other hand HIF-1alpha protein is selectively required for down-regulation of ASK1 activated during LPS-induced TLR4 downstream signalling.

Involvement of hypoxia-inducible factor-1 HiF(1α) in IgE-mediated primary human basophil responses

Basophils play a pivotal role in regulating chronic allergic inflammation as well as angiogenesis. Here, we show for the first time that IgE‐mediated activation of primary human basophils results in protein accumulation of the α‐subunit of hypoxia‐inducible factor 1α (HIF‐1α), which is differentially regulated compared with signals controlling histamine release. HIF‐1 facilitates cellular adaptation to hypoxic conditions such as inflammation and tumour growth by controlling glycolysis, angiogenesis and cell adhesion. ERK and p38 MAPK, but not reactive oxygen species (ROS), ASK1 or PI 3‐kinase, were critical for IgE‐mediated accumulation of HIF‐1α, although the latter crucially affected degranulation. Abrogating HIF‐1α expression in basophils using siRNA demonstrated that this protein is essential for vascular endothelial growth factor (VEGF) mRNA expression and, consequently, release of VEGF protein. In addition, HIF‐1α protein alters IgE‐induced ATP depletion in basophils, thus also supporting the production of the pro‐allergic cytokine IL‐4.

A novel pesticide-induced conformational state of the oestrogen receptor ligand-binding domain, detected by conformation-specific peptide binding

The diverse effects of different natural and synthetic oestrogen receptor ligands depend on induction of different receptor conformations, allowing differential interactions with other transcription factors. Different conformations of the oestrogen receptor ligand binding domains can be monitored by conformation‐specific binding to peptides selected from phage‐displayed peptide libraries. We now report that a group of chlorinated pesticides, including 2,4‐dichlorodiphenyl‐dichloroethylene, induces a peptide recognition pattern different from those induced by any one of the classical oestrogen receptor ligands. The pesticide‐complexed oestrogen receptors recognized peptides reacting with the receptors complexed both with the natural oestrogen 17β‐oestradiol and with the synthetic partial antagonist 4‐hydroxy‐tamoxifen, respectively, indicating that the pesticide‐induced conformation shares features with both the 17β‐oestradiol‐ and the 4‐hydroxy‐tamoxifen‐induced conformations. The substitution H524A in the ligand binding domain conferred the pesticide‐specific peptide recognition pattern and transactivation activity to the oestradiol‐ and the 4‐hydroxy‐tamoxifen‐complexed receptors, indicating that one important determinant of the pesticide‐induced conformation is a lack of stabilisation of any one particular receptor conformation by ligand interaction with H524, which is known to interact with both oestradiol and 4‐hydroxy‐tamoxifen. Thus, peptide binding analyses of oestrogen receptor conformations induced by environmental endocrine disruptors can give novel information about molecular mechanisms of oestrogen action in general.

Involvement of Hypoxia-Inducible Factor-1 in the Inflammatory Responses of Human LAD2 Mast Cells and Basophils

We recently showed that hypoxia-inducible factor 1 (HIF-1) plays a crucial role in the pro-allergic functions of human basophils by transcriptional control of energy metabolism via glycolysis as well as directly triggering expression of the angiogenic cytokine vascular endothelium growth factor (VEGF). Here, we investigated HIF-1 involvement in controlling the synthesis of angiogenic and inflammatory cytokines from various human effector cells stimulated by IgE-dependent or innate immune triggers. Purified primary human basophils, LAD2 human mast cells and THP-1 human myeloid cells were used for investigations of FcεRI and Toll-like receptor (TLR) ligand-induced responses. In contrast to basophils, LAD2 mast cells expressed background levels of HIF-1α, which was largely independent of the effects of stem cell factor (SCF). Both mast cells and basophils expressed TLR2 and 4, albeit weakly compared to THP-1 cells. Cytokine production in mast cells following TLR ligand stimulation was markedly reduced by HIF-1α knockdown in LAD2 mast cells. In contrast, although HIF-1 is involved in IgE-mediated IL-4 secretion from basophils, it is not clearly induced by peptidoglycan (PGN). HIF-1α accumulation is critical for sustaining human allergic effector cell survival and function. This transcription complex facilitates generation of both pro-angiogenic and inflammatory cytokines in mast cells but has a differential role in basophil stimulation comparing IgE-dependent triggering with innate immune stimuli.