GAPDH delivers heme to soluble guanylyl cyclase

Soluble guanylyl cyclase (sGC) is a key component of NO–cGMP signaling in mammals. Although heme must bind in the sGC β1 subunit (sGCβ) for sGC to function, how heme is delivered to sGCβ remains unknown. Given that GAPDH displays properties of a heme chaperone for inducible NO synthase, here we investigated whether heme delivery to apo-sGCβ involves GAPDH. We utilized an sGCβ reporter construct, tetra-Cys sGCβ, whose heme insertion can be followed by fluorescence quenching in live cells, assessed how lowering cell GAPDH expression impacts heme delivery, and examined whether expressing WT GAPDH or a GAPDH variant defective in heme binding recovers heme delivery. We also studied interaction between GAPDH and sGCβ in cells and their complex formation and potential heme transfer using purified proteins. We found that heme delivery to apo-sGCβ correlates with cellular GAPDH expression levels and depends on the ability of GAPDH to bind intracellular heme, that apo-sGCβ associates with GAPDH in cells and dissociates when heme binds sGCβ, and that the purified GAPDH–heme complex binds to apo-sGCβ and transfers its heme to sGCβ. On the basis of these results, we propose a model where GAPDH obtains mitochondrial heme and then forms a complex with apo-sGCβ to accomplish heme delivery to sGCβ. Our findings illuminate a critical step in sGC maturation and uncover an additional mechanism that regulates its activity in health and disease.

Glycolytic Biomarkers Predictive of Transformation in Patients with Follicular Lymphoma

Introduction Follicular lymphoma (FL) is an indolent lymphoma derived from germinal center B cells, covering approximately 20% of all lymphomas. The malignancy is generally considered an incurable condition, hence with a median survival time exceeding 10 years. A portion of patients experience early progression or histological transformation (HT) to a more aggressive lymphoma, typically diffuse large B cell lymphoma which occurs in up to 45% of FL patients, reducing the median survival after transformation to 1-2 years. Early detection of reliable predictors of HT would allow pre-emptive therapeutic intervention strategies and aim at improved survival for patients with transformed FL. In this study, we focus on the two glycolytic enzymes, Aldolase A and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Both proteins have previously been found to be upregulated at diagnosis in various solid cancers and associated with poor survival. In addition to its glycolytic effects, GAPDH has been recognized for its non-glycolytic effects including regulation of DNA replication and repair, RNA nuclear export and apoptosis. Aim The aim of this study was to identify biomarkers predictive of HT in patients with FL, by investigating the correlation between intratumoral aldolase A and GAPDH expression levels with the risk of transformation in pre-therapeutic tumor samples from FL patients. Materials and Methods Immunohistochemical aldolase A and GAPDH expression levels were quantified using digital image analysis in pre-therapeutic tumor tissue from FL patients at time of diagnosis for patients without (n=51) or with (n=41) subsequent HT, as well as in sequential samples at time of transformation (n=41). Staining intensities were assessed as area fractions (AFs) of the stained area normalized to the region of interest on whole biopsy sections. Results At time of initial FL diagnosis, FL patients with subsequent transformation had significantly higher levels of aldolase A and GAPDH expression compared to patients with no subsequent transformation, (p<0.001 and p=0.005). High levels of aldolase A expression (75th percentile cut-off), were found to be associated with a significantly shorter transformation free survival (TFS) (p=0.015) and progression free survival (PFS) (p=0.001). Furthermore, high levels of GAPDH expression were found to be associated with a significantly shorter TFS (p<0.001), PFS (p=0.034) as well as overall survival (OS) (p=0.018). Conclusion These results suggest that at time of initial FL diagnosis, aldolase A expression and GAPDH expression, maybe as indicators of high metabolic turnover, may be predictive of the patient's risk of subsequent histological transformation. Disclosures No relevant conflicts of interest to declare.

Changes in the expression of galanin and galanin receptors in the wall of the colon in pigs experimentally infected with Brachyspira hyodysenteriae

The expression of galanin (GAL) and its three receptors (GalR1, GalR2, and GalR3) were studied with real-time PCR in the colonic wall of pigs suffering from experimental colitis caused by the infection with Brachyspira hyodysenteriae. The expression was studied in the muscular membrane, mucosa/submucosa layer, and in lymphocytes isolated from mucosa/submucosa. The expression levels were normalized to glyceraldehyde-6-phosphate dehydrogenase (GAPDH) expression and compared to expression levels in control animals. GAL expression was found in all three studied compartments of the colonic wall. A significant decrease in GAL expression level was found in the mucosa/submucosa and in isolated lymphocytes, whereas the decrease was much less profound in the muscular membrane. In the case of galanin receptors their expression was found in all studied compartments of the colonic wall, however at different levels, as compared to GAPDH expression. The decrease of galanin receptors expression was found in all studied compartments of the colonic wall of the sick animals.