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Journal of Molecular Endocrinology Oct 2012We have previously demonstrated that a homozygous inactivating P86S mutation of the glucagon receptor (GCGR) causes a novel human disease of hyperglucagonemia,...
We have previously demonstrated that a homozygous inactivating P86S mutation of the glucagon receptor (GCGR) causes a novel human disease of hyperglucagonemia, pancreatic α-cell hyperplasia, and pancreatic neuroendocrine tumors (Mahvash disease). The mechanisms for the decreased activity of the P86S mutant (P86S) are abnormal receptor localization to the endoplasmic reticulum (ER) and defective interaction with glucagon. To search for targeted therapies for Mahvash disease, we examined whether P86S can be trafficked to the plasma membrane by pharmacological chaperones and whether novel glucagon analogs restore effective receptor interaction. We used enhanced green fluorescent protein-tagged P86S stably expressed in HEK 293 cells to allow fluorescence imaging and western blotting and molecular modeling to design novel glucagon analogs in which alanine 19 was replaced with serine or asparagine. Incubation at 27 °C largely restored normal plasma membrane localization and normal processing of P86S but osmotic chaperones had no effects. The ER stressors thapsigargin and curcumin partially rescued P86S. The lipophilic GCGR antagonist L-168,049 also partially rescued P86S, so did Cpd 13 and 15 to a smaller degree. The rescued P86S led to more glucagon-stimulated cAMP production and was internalized by glucagon. Compared with the native glucagon, the novel glucagon analogs failed to stimulate more cAMP production by P86S. We conclude that the mutant GCGR is partially rescued by several pharmacological chaperones and our data provide proof-of-principle evidence that Mahvash disease can be potentially treated with pharmacological chaperones. The novel glucagon analogs, however, failed to interact with P86S more effectively.
Topics: Alanine; Asparagine; Cell Membrane; Curcumin; Cyclic AMP; Drug Design; Glucagon; Green Fluorescent Proteins; HEK293 Cells; Humans; Molecular Chaperones; Mutation; Pancreatic Neoplasms; Protein Transport; Pyridines; Pyrroles; Receptors, Glucagon; Serine; Thapsigargin
PubMed: 22693263
DOI: 10.1530/JME-12-0051 -
Endocrinologia Y Nutricion : Organo de... 2011To elucidate the pathogenetic mechanisms of a mutant P86S glucagon receptor (GCGR) in causing a novel human disease (Mahvash disease).
BACKGROUND AND AIM
To elucidate the pathogenetic mechanisms of a mutant P86S glucagon receptor (GCGR) in causing a novel human disease (Mahvash disease).
MATERIAL AND METHOD
Enhanced green fluorescent protein (EGFP)-tagged WT and P86S GCGR were expressed in HEK 293 or H1299 cells either transiently or stably. Receptor localization and internalization, and cell apoptosis were studied by fluorescence microscopy, and calcium signaling by Rhod-3 labeling. Gene expression was assayed by RT-PCR or Western blot. Cell fate was determined by live cell imaging.
RESULTS
Unlike WT GCGR, P86S was partially localized to the plasma membrane and partially in the cytoplasm as previously reported and did not undergo internalization upon glucagon treatment. P86S did not elicit calcium response after treatment with 1 μM glucagon. Cells transiently expressing P86S exhibited more apoptosis than those expressing WT GCGR (18.3% vs 2.1%, P<0.05) but the X-box binding protein 1 mRNA cleavage, a marker of endoplasmic reticulum (ER) stress, was not evident, suggesting that the apoptosis did not result from ER stress. Cells stably expressing P86S did not exhibit apoptosis and a quarter of them harbored a novel inclusion body-like circular structure that was marked by P86S and ER residential proteins. These circular ER bodies were not seen in cells expressing WT GCGR or transiently expressing P86S and were not affected by treatment with proteasome inhibitor or microtubule depolymerizer, suggesting that they do not represent aggresome structures. The circular ER bodies could fuse and split to form new bodies.
CONCLUSION
The naturally-occurring P86S mutant GCGR exhibits abnormal receptor internalization and calcium mobilization, and causes apoptosis. The novel dynamic circular ER bodies may be adaptive in nature to nullify the toxic effects on P86S. These findings provide further insights into the pathogenetic mechanisms of Mahvash disease.
Topics: Apoptosis; Calcium Signaling; Cell Line; Cell Membrane; Cyclic AMP; Endocytosis; Endoplasmic Reticulum; Glucagon; Glucagon-Secreting Cells; Humans; Hyperplasia; Hypoglycemia; Mutation, Missense; Neoplastic Syndromes, Hereditary; Neuroendocrine Tumors; Pancreatic Neoplasms; Point Mutation; Receptors, Glucagon; Recombinant Fusion Proteins
PubMed: 21680267
DOI: 10.1016/j.endonu.2011.04.002 -
Biology of Blood and Marrow... Oct 2011BK virus (BKV) is a human polyomavirus that remains latent in the urinary tract epithelium in most individuals. However, in immunocompromised states, including after... (Clinical Trial)
Clinical Trial
BK virus (BKV) is a human polyomavirus that remains latent in the urinary tract epithelium in most individuals. However, in immunocompromised states, including after hematopoietic stem cell transplantation (HSCT), BKV may reactivate and cause infection predominantly affecting the bladder, commonly manifested as hemorrhagic cystitis. Renal insufficiency, occasionally requiring hemodialysis, is not uncommon and was previously attributed to medications or the development of tubulointestitial nephritis. We report a series of 6 HSCT recipients who developed obstructive uropathy of the upper urinary tract system secondary to inflammation and hemorrhage involving the upper uroepithelium, causing ureteral stenosis. Temporary placement of a percutaneous nephrostomy catheter relieved the obstruction and significantly improved kidney function, successfully preventing progression to more advanced renal disease in these patients.
Topics: BK Virus; Female; Hematopoietic Stem Cell Transplantation; Humans; Immunocompromised Host; Male; Nephrostomy, Percutaneous; Polyomavirus Infections; Transplantation, Homologous; Ureteral Obstruction; Urinary Catheterization
PubMed: 21396475
DOI: 10.1016/j.bbmt.2011.03.002 -
IEEE Transactions on Information... Jul 2006Many minimally invasive surgeries (MISs) involve removing whole organs or tumors that are connected to other organs. Development of haptic simulators that reproduce...
Many minimally invasive surgeries (MISs) involve removing whole organs or tumors that are connected to other organs. Development of haptic simulators that reproduce separation forces between organs can help surgeons learn MIS procedures. Powerful computational approaches such as finite-element methods generally cannot simulate separation in real time. This paper presents a novel approach for real-time computation of separation forces between deformable bodies. Separation occurs either due to fracture when a tool applies extensive forces to the bodies or due to evaporation when a laser beam burns the connection between the bodies. The separation forces are generated online from precalculated force-displacement functions that depend on the local adhesion/separation states between bodies. The precalculated functions are accurately synthesized from a large number of force responses obtained through either offline simulation, measurement, or analytical approximation during the preprocessing step. The approach does not require online computation of force versus global deformation to obtain separation forces. Only online interpolation of precalculated responses is required. The state of adhesion/separation during fracture and evaporation are updated by computationally simple models, which are derived based on the law of conservation of energy. An implementation of the approach for the haptic simulation of the removal of a diseased organ is presented, showing the fidelity of the simulation.
Topics: Animals; Computer Simulation; Elasticity; Humans; Models, Biological; Physical Stimulation; Stress, Mechanical; Surgery, Computer-Assisted; Touch; User-Computer Interface
PubMed: 16871732
DOI: 10.1109/titb.2006.872040