Kathleen I. J. Shennan

PROCESSING OF PRO-ISLET AMYLOID POLYPEPTIDE (PROIAPP) BY THE PROHORMONE CONVERTASE PC2

Islet amyloid polypeptide (IAPP), ‘amylin’, is the component peptide of islet amyloid formed in Type 2 diabetes. IAPP is expressed in islet β‐cells and is derived from a larger precursor, proIAPP, by proteolysis. An in vitro translation/translocation system was used to separately examine processing of human proIAPP by the β‐cell endopeptidases PC2, PC3 or furin. ProIAPP was converted to mature IAPP by PC2 but there was little conversion by furin or PC3. These data are consistent with processing of proIAPP in β‐cell secretory granules. Abnormal cellular proteolysis associated with type 2 diabetes could contribute to IAPP amyloidosis.

Inhibitory effect of Pax4 on the human insulin and islet amyloid polypeptide (IAPP) promoters

Pax4 is a paired‐box transcription factor that plays an important role in the development of pancreatic β‐cells. Two Pax4 cDNAs were isolated from a rat insulinoma library. One contained the full‐length sequence of Pax4. The other, termed Pax4c, was identical to Pax4 but lacked the sequences encoding 117 amino acids at the COOH‐terminus. Pax4 was found to inhibit the human insulin promoter through a sequence element, the C2 box, located at −253 to −244, and the islet amyloid polypeptide promoter through a sequence element located downstream of −138. The inhibitory activity of Pax4 was mapped to separate regions of the protein between amino acids 2–230 and 231–349.

Signal peptide mutations in RANK prevent downstream activation of NF-κB

Familial expansile osteolysis and related disorders are caused by heterozygous tandem duplication mutations in the signal peptide region of the gene encoding receptor activator of NF‐κB (RANK), a receptor critical for osteoclast formation and function. Previous studies have shown that overexpression of these mutant proteins causes constitutive activation of NF‐κB signaling in vitro, and it has been assumed that this accounts for the focal osteolytic lesions that are seen in vivo. We show here that constitutive activation of NF‐κB occurred in HEK293 cells overexpressing wild‐type or mutant RANK but not in stably transfected cell lines expressing low levels of each RANK gene. Importantly, only cells expressing wild‐type RANK demonstrated ligand‐dependent activation of NF‐κB. When overexpressed, mutant RANK did not localize to the plasma membrane but localized to extensive areas of organized smooth endoplasmic reticulum, whereas, as expected, wild‐type RANK was detected at the plasma membrane and in the Golgi apparatus. This intracellular accumulation of the mutant proteins is probably the result of lack of signal peptide cleavage because, using two in vitro translation systems, we demonstrate that the mutations in RANK prevent cleavage of the signal peptide. In conclusion, signal peptide mutations lead to accumulation of RANK in the endoplasmic reticulum and prevent direct activation by RANK ligand. These results strongly suggest that the increased osteoclast formation/activity caused by these mutations cannot be explained by studying the homozygous phenotype alone but requires further detailed investigation of the heterozygous expression of the mutant RANK proteins.