Impact of Extrinsic and Intrinsic Hypoxia on Catecholamine Biosynthesis in Absence or Presence of Hif2α in Pheochromocytoma Cells

Pheochromocytomas and paragangliomas (PPGLs) with activated pseudohypoxic pathways are associated with an immature catecholamine phenotype and carry a higher risk for metastasis. For improved understanding of the underlying mechanisms we investigated the impact of hypoxia and pseudohypoxia on catecholamine biosynthesis in pheochromocytoma cells naturally lacking Hif2α (MPC and MTT) or expressing both Hif1α and Hif2α (PC12). Cultivation under extrinsic hypoxia or in spheroid culture (intrinsic hypoxia) increased cellular dopamine and norepinephrine contents in all cell lines. To distinguish further between Hif1α- and Hif2α-driven effects we expressed Hif2α in MTT and MPC-mCherry cells (naturally lacking Hif2α). Presence of Hif2α resulted in similarly increased cellular dopamine and norepinephrine under hypoxia as in the control cells. Furthermore, hypoxia resulted in enhanced phosphorylation of tyrosine hydroxylase (TH). A specific knockdown of Hif1α in PC12 diminished these effects. Pseudohypoxic conditions, simulated by expression of Hif2α under normoxia resulted in increased TH phosphorylation, further stimulated by extrinsic hypoxia. Correlations with PPGL tissue data led us to conclude that catecholamine biosynthesis under hypoxia is mainly mediated through increased phosphorylation of TH, regulated as a short-term response (24-48 h) by HIF1α. Continuous activation of hypoxia-related genes under pseudohypoxia leads to a HIF2α-mediated phosphorylation of TH (permanent status).Funding: This research was funded by the Deutsche Forschungsgemeinschaft (DFG) within the CRC/Transregio205/1 (project number: 314061271-TRR 205), Project No. B12 (N.B. and G.E.), Project No. B10 (S.R., J.P. and M.U.)and Project No. S01 (A.W., C.G. and M.P.) “The Adrenal: Central Relay in Health and Disease“, and by theParadi erence Foundation (N.B., I.P., S.R. and G.E.).S

Three-Dimensional Modelling of Honeybee Venom Allergenic Proteases: Relation to Allergenicity

Api SI and Api SIT are serine proteases of the honeybee venom containing allergenic determinants. Each protease consists of two structural modules: an N-terminal CUB (Api SI) or a clip domain (Api SII) and a C-terminal serine protease-like (SPL) domain. Both domains are connected with a linker peptide. The knowledge about the structure and function of Api SI and Api SII is limited mainly to their amino acid sequences. We constructed 3-D models of the two proteases using their amino acid sequences and crystallographic coordinates of related proteins. The models of the SPL domains were built using the structure of the prophenoloxidase-activating factor (PPAF)-II as a template. For modelling of the Api SI CUB domain the coordinates of porcine spermadhesin PSP-I were used. The models revealed the catalytic and substrate-binding sites and the negatively charged residue responsible for the trypsin-like activity. IgE-binding and antigenic sites in the two allergens were predicted using the models and programs based on the structure of known epitopes. Api SI and Api SII show structural and functional similarity to the members of the PPAF-II family. Most probably, they are part of the defence system of Apis mellifera.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Mass spectrometry imaging identifies metabolic patterns associated with malignant potential in pheochromocytoma and paraganglioma

Objective Within the past decade, important genetic drivers of pheochromocytoma and paraganglioma (PPGLs) development have been identified. The pathophysiological mechanism that translates these alterations into functional autonomy and potentially malignant behavior has not been elucidated in detail. Here we used MALDI-mass spectrometry imaging (MALDI-MSI) of formalin-fixed paraffin-embedded tissue specimens to comprehensively characterize the metabolic profiles of PPGLs. Design and methods MALDI-MSI was conducted in 344 PPGLs and results correlated with genetic and phenotypic information. We experimentally silenced genetic drivers by siRNA in PC12 cells to confirm their metabolic impact in vitro. Results Tissue abundance of kynurenine pathway metabolites such as xanthurenic acid was significantly lower (P = 2.35E-09) in the pseudohypoxia pathway cluster 1 compared to PPGLs of the kinase-driven PPGLs cluster 2. Lower abundance of xanthurenic acid was associated with shorter metastasis-free survival (log-rank tests P = 7.96E-06) and identified as a risk factor for metastasis independent of the genetic status (hazard ratio, 32.6, P = 0.002). Knockdown of Sdhb and Vhl in an in vitro model demonstrated that inositol metabolism and sialic acids were similarly modulated as in tumors of the respective cluster. Conclusions The present study has identified distinct tissue metabolomic profiles of PPGLs in relation to tumor genotypes. In addition, we revealed significantly altered metabolites in the kynurenine pathway in metastatic PPGLs, which can aid in the prediction of its malignant potential. However, further validation studies will be required to confirm our findings