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Impaired NH₂-Terminal Processing of Human Proislet Amyloid Polypeptide by the Prohormone Convertase PC2 Leads to Amyloid Formation and Cell Death

¹ Department of Pathology and Laboratory Medicine and Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada
² Pacific Northwest Research Institute, University of Washington, Seattle, Washington
³ Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois
⁴ Larry Hillblom Islet Research Center, University of California, Los Angeles, California
⁵ Department of Genetic Medicine and Development, University Medical Center, Geneva, Switzerland

Address correspondence and reprint requests to Dr. Lucy Marzban, Child and Family Research Institute, Rm. 2071-950 West 28th Ave., Vancouver, BC, Canada V5Z 4H4. E-mail: marzban{at}interchange.ubc.ca

Abbreviations: EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; GFP, green fluorescent protein; IAPP, islet amyloid polypeptide; MOI, multiplicity of infection; PC1/3, prohormone convertase 1/3; PC2, prohormone convertase 2; TUNEL, transferase-mediated dUTP nick-end labeling

Islet amyloid, formed by aggregation of islet amyloid polypeptide (IAPP; amylin), is a pathological characteristic of the pancreas in type 2 diabetes and may contribute to the progressive loss of ß-cells in this disease. We tested the hypothesis that impaired processing of the IAPP precursor proIAPP contributes to amyloid formation and cell death. GH3 cells lacking the prohormone convertase 1/3 (PC1/3) and IAPP and with very low levels of prohormone convertase 2 (PC2) were transduced with adenovirus (Ad) expressing human or rat (control) proIAPP linked to green fluorescent protein, with or without Ad-PC2 or Ad-PC1/3. Expression of human proIAPP increased the number of transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells 96 h after transduction (+hIAPP 8.7 ± 0.4% vs. control 3.0 ± 0.4%; P < 0.05). COOH-terminal processing of human proIAPP by PC1/3 increased (hIAPP+PC1/3 10.4 ± 0.7%; P < 0.05), whereas NH₂-terminal processing of proIAPP by addition of PC2 markedly decreased (hIAPP+PC2 5.5 ± 0.5%; P < 0.05) the number of apoptotic GH3 cells. Islets from mice lacking PC2 and with ß-cell expression of human proIAPP (hIAPP^+/+/PC2^–/–) developed amyloid associated with ß-cell death during 2-week culture. Rescue of PC2 expression by ex vivo transduction with Ad-PC2 restored NH₂-terminal processing to mature IAPP and decreased both the extent of amyloid formation and the number of TUNEL-positive cells (–PC2 26.5 ± 4.1% vs. +PC2 16.1 ± 4.3%; P < 0.05). These findings suggest that impaired NH₂-terminal processing of proIAPP leads to amyloid formation and cell death and that accumulation of the NH₂-terminally extended human proIAPP intermediate may be a critical initiating step in amyloid formation.

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				L. Marzban, A. Tomas, T. C. Becker, L. Rosenberg, J. Oberholzer, P. E. Fraser, P. A. Halban, and C. B. Verchere Small Interfering RNA-Mediated Suppression of Proislet Amyloid Polypeptide Expression Inhibits Islet Amyloid Formation and Enhances Survival of Human Islets in Culture Diabetes, November 1, 2008; 57(11): 3045 - 3055. [Abstract] [Full Text] [PDF]