Signal sequence mutation in autosomal dominant form of hypoparathyroidism induces apoptosis that is corrected by a chemical chaperone

Abstract

Autosomal dominant familial isolated hypoparathyroidism (AD-FIH) is caused by a Cys 3 Arg mutation (C18R) in the hydrophobic core of the signal peptide of human preproparathyroid hormone (PPTH). Although this mutation impairs secretion of the hormone, the mechanism by which one mutant allele produces the autosomal-dominant disease is unexplained. Using transfected HEK293 cells, we demonstrate that the expressed mutant hormone is trapped intracellularly, predominantly in the endoplasmic reticulum (ER). This ER retention was found to be toxic for the cells, which underwent apoptosis, as evident from the marked increase in the number of cells staining positive for Annexin V binding and for the TUNEL reaction. The cells producing mutant hormone also had marked up-regulation of the ER stress-responsive proteins, BiP and PERK, as well as the proapoptotic transcription factor, CHOP. Up-regulation of these markers of the unfolded protein response supported a causal link between the ER stress and the cell death cascade. When the C18R PPTH was expressed in the presence of 4-phenylbutyric acid, which is a pharmacological chaperone, intracellular accumulation was reduced and normal secretion was restored. This treatment also produced remarkable reduction of ER stress signals and protection against cell death. These data implicate ER stress-induced cell death as the underlying mechanism for AD-FIH and suggest that the pharmacological manipulation of this pathway by using chemical chaperones offers a therapeutic option for treating this disease.endoplasmic reticulum stress ͉ parathyroid hormone ͉ unfolded protein response ͉ 4-phenylbutyric acid F amilial isolated hypoparathyroidism (FIH) represents a heterogeneous group of genetic disorders characterized by low calcium and high phosphorus levels in the blood serum, where the amount of immunoreactive parathyroid hormone (PTH) is low or undetectable. Clinical symptoms include neuromuscular irritability, tingling of the fingers and toes, and spontaneous or latent tetany (1, 2). These diseases vary in mode of inheritance. Mutations in the preproparathyroid hormone (PPTH) gene are found to be associated with the disease in some pedigrees (3-5).PPTH is synthesized as a 115-aa precursor in the parathyroid gland and is converted to the pro-PTH upon removal of the signal peptide (by signal peptidase) within the endoplasmic reticulum (ER). After trafficking to the Golgi apparatus, the pro-PTH is cleaved to the 84-aa mature PTH (Fig. 1). Then the mature PTH is packaged into secretory granules and, depending on needs, released into the blood, where it functions as a major regulator of the calcium level. The release of the PTH by the parathyroid gland is strictly calcium-dependent (6).In 1990, Arnold et al. (3) identified a T to C point mutation in the signal peptide-encoding region of the PPTH gene in a family with autosomal dominant FIH (AD-FIH) (7). This mutation disrupts the hydrophobic core of the signal sequence by changing the codon at position 18 (Ϫ8 position with respect to the s...

Endoplasmic Reticulum Stress-induced Apoptosis in Leishmania through Ca2+-dependent and Caspase-independent Mechanism

“…Chemical chaperone, such as PBA, is a low molecular weight compound known to stabilize protein conformation, improve ER folding capacity, and facilitate the trafficking of mutant proteins (48,49). To investigate the action of chemical chaperone, we first tested whether PBA protected against TM-induced ER stress as well as DNA fragmentation and PS externalization in Leishmania.…”

Pathogenesis of retinitis pigmentosa associated with apoptosis-inducing mutations in carbonic anhydrase IV

“…Thus, sustained accu- mulation of both unfolded proteins exceeded the threshold of ER stress required to induce apoptosis. ER stress-induced apoptosis has been implicated in several human disorders, including diabetes, Parkinson's disease, Alzheimer's disease, hypoparathyroidism, and also in 1 form of RP caused by the P23H rhodopsin mutation (17)(18)(19)(20)(21). That ER stress may be a pathogenic mechanism underlying retinal degeneration was also suggested by Colley et al, who found that NinaA, a photoreceptor specific homologue of cyclophilin, was required for export of Rh1 rhodopsin from the ER.…”

“…COS-7 and HEK-293 cells (American Type Culture Collection) were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 2 mM L-glutamine and 10% heat-inactivated FBS in a humidified atmosphere of 5% CO 2 at 37°C. The transfection procedure is as described earlier (20). All experiments were performed at 72-h post-transfection.…”

“…Immunocytochemistry was performed following previously described procedure (20). Briefly, cells were fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100 (for cell surface-labeling experiments, the permeabilzation step was omitted).…”

“…Thermal cycling conditions were: 95°C for 5 min; and 35 cycles of 95°C/45 s, 60°C/45 s, and 68°C/45 s. All reactions were carried out in triplicate. The relative transcript level of the target gene was calculated as previously described (20).…”