IRAK3 modulates downstream innate immune signalling through its guanylate cyclase activity

Interleukin-1 receptor associated kinase 3 (IRAK3) is a cytoplasmic homeostatic mediator of inflammatory responses and is potentially useful as a prognostic marker in inflammation. IRAK3 inhibits signalling cascades downstream of myddosome complexes associated with toll like receptors. IRAK3 contains a death domain that interacts with other IRAK family members, a pseudokinase domain and a C-terminus domain involved with tumour necrosis factor receptor associated factor 6 (TRAF6). Previous bioinformatic studies revealed that IRAK3 contained a guanylate cyclase centre in its pseudokinase domain but its role in IRAK3 action is unresolved. We demonstrate that wildtype IRAK3 is capable of producing cGMP. Furthermore, we show that a specific point mutation in the guanylate cyclase centre reduced cGMP production. Cells containing toll like receptor 4 and a nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB) reporter system were transfected with IRAK3 or mutant IRAK3 proteins. Cell-permeable cGMP treatment of untransfected control cells suppresses downstream signalling through modulation of the NFĸB in the presence of lipopolysaccharides. Cells transfected with wildtype IRAK3 also suppress lipopolysaccharide induced NFĸB activity in the absence of exogenous cGMP. Lipopolysaccharide induced NFĸB activity was not suppressed in cells transfected with the IRAK3 mutant with reduced cGMP-generating capacity. Whereas in the presence of exogenously applied cell-permeable cGMP the IRAK3 mutant was able to retain its function by suppressing lipopolysaccharide induced NFĸB activity. Furthermore, increasing the amount of membrane permeable cGMP did not affect IRAK3’s ability to reduce NFĸB activity. These results suggest that cGMP generated by IRAK3 may be involved in regulatory function of the protein where the presence of cGMP may selectively affect downstream signalling pathway(s) by modulating binding and/or activity of nearby proteins that interact in the inflammatory signalling cascade

Calcium is the molecular switch shifting the phytosulfokine receptor 1 (PSKR1) from kinase to guanylate cyclase activity

Conference presentationMany plant responses are mediated by interactions between intracellular calcium and the second messenger cGMP formed by guanylate cyclases (GCs). Previously we identified a novel class of receptor-GCs containing the GC catalytic center embedded within the kinase domain and showed that the recombinant cytoplasmic domain of phytosulfokine receptor AtPSKR1 has both guanylate cyclase and kinase activity in vitro (Kwezi et al. 2011 J Biol Chem 286: 22580-8). We now show that physiological increases in calcium levels enhance GC activity of AtPSKR1 whereas these calcium levels reversibly inhibit kinase activity. In addition PSKR1 kinase activity is reduced in the presence of the GC product cGMP. Recombinant AtPSKR1 can undergo in vitro autophosphorylation and we have confirmed it has 14 phosphorylation sites in its cytoplasmic domain including 8 serine, 3 threonine and 3 tyrosine residues. Three phospho-serine residues at the juxta-membrane position were mutated to either mimic phosphorylation on or off states. Kinase activity was enhanced in the on mutant and suppressed in the off mutant while GC activity was unaffected suggesting calcium acts as a molecular switch of PSKR1- mediated signalling that can be modulated by the phosphorylation state. The challenge now lies in understanding how molecular interactions between the GC and kinase domains are capitalized on in the plant

Burnout and Occupational Stress Among Hungarian Radiographers Working in Emergency and Non-Emergency Departments During COVID-19 Pandemic

Introduction: The increased workload caused by the coronavirus pandemic may have had a significant impact on the mental health of radiographers. The aim of our study was to investigate burnout and occupational stress in radiographers working in emergency departments (ED) and non-emergency departments (NED). Methods: Quantitative, cross-sectional, descriptive research was carried out among radiographers working in the public health sector in Hungary. Due to the cross-sectional nature of our survey, there was no overlap between the ED and NED groups. For data collection, we used simultaneously the Maslach Burnout Inventory (MBI), the Effort-Reward Imbalance questionnaire (ERI), and our self-designed questionnaire. Results: We excluded incomplete questionnaires from our survey; finally, 439 responses were evaluated. Significantly higher scores for depersonalisation (DP; 8.43 (SD = 6.69) vs. 5.63 (SD = 4.21) and emotional exhaustion (EE; 25.07 (SD = 11.41) vs. 19.72 (SD = 11.72)) were observed in radiographers working in ED (p = 0.001; p = 0.001) when compared to NED. Male radiographers working in ED aged 20–29 and 30–39 years with experience of 1–9 years were more affected by DP (p ≤ 0.05). Worrying about one's own health had a negative effect on DP and EE (p ≤ 0.05). Having close friend with a COVID-19 infection had a negative effect on EE (p ≤ 0.05); not being infected with coronavirus, not being quarantined and relocating within the workplace had a positive effect on personal accomplishment (PA); radiographers who were 50 years or older with 20–29 years of experience were more affected by depersonalisation (DP); and those who worried about their health had significantly higher stress scores (p ≤ 0.05) in both ED and NED settings. Conclusion: Male radiographers at the beginning of their careers were more affected by burnout. Employment in EDs had a negative impact on DP and EE. Implications for practice: Our results support the implementation of interventions to counter the effects of occupational stress and burnout among radiographers working in ED