Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Apr 8;280(14):13209-12.
doi: 10.1074/jbc.C400475200. Epub 2005 Feb 10.

Proinsulin disulfide maturation and misfolding in the endoplasmic reticulum

Ming Liu  1 , Yulin Li, Douglas Cavener, Peter Arvan
Affiliations

Proinsulin disulfide maturation and misfolding in the endoplasmic reticulum

Ming Liu et al. J Biol Chem. .

Abstract

Upon nonreducing Tris-Tricine-urea-SDS-PAGE, newly synthesized proinsulin from pancreatic islets of normal rodents forms a band fast mobility representing the native disulfide isomer, which is efficiently secreted. In addition at least two slower migrating "isomer 1 and 2" bands are recovered, not discernible under reducing conditions, which represent minor species that exhibit less efficient secretion. Although rats and mice have two proinsulin genes, three distinct migrating species are also produced upon proinsulin expression from a single wild-type human proinsulin cDNA. The "Akita-type" proinsulin mutation, which causes dominant-negative diabetes mellitus due to point mutation C(A7)Y that leaves B7-cysteine without its disulfide pairing partner, is recovered as a form that near quantitatively co-migrates with the aberrant isomer 1 band of proinsulin. Anomalous migration is also demonstrated for several other mutants lacking a single cysteine. In islets from PERK-/- mice, which exhibit premature loss of pancreatic beta cells, hypersynthesis of proinsulin increases the amount of nonnative proinsulin isomers. Such findings appear consistent with an hypothesis that supranormal production of nonnative proinsulin may predispose to pancreatic beta cell toxicity.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1. Newly synthesized proinsulin in rat pancreatic islets analyzed by nonreducing Tris-Tricine-urea-SDS-PAGE
Isolated rat islets were divided into distinct samples that were radiolabeled with 35S-labeled amino acid mixture for the times indicated and then lysed immediately or after a 30-min chase period. Fifty islets were used for each sample. The cell lysates and 30-min chase medium were immunoprecipitated with a polyclonal antibody to insulin that recognizes all insulin forms. The immunoprecipitates were analyzed by nonreducing Tris-Tricine-urea-SDS-PAGE. In addition to the established positions of proinsulin (Pro), insulin (Ins), and conversion intermediates (bracket), two proinsulin bands migrating more slowly than native proinsulin were identified (double arrows marked with asterisk), and one of these forms appears to be co-precipitated with anti-BiP.
FIG. 2
FIG. 2. Recombinant proinsulin synthesized from the human proinsulin cDNA in heterologous cells
A, forty-eight hours after transfection (Proins) or mock-transfection (Con) of the CLA14 subclone of CHO cells (13), the cells were pulse-labeled with 35S-labeled amino acids for 1 h and chased for 50 min. The cells (“C”) were lysed and chase media (“M”) collected, and both were analyzed by Tris-Tricine-urea-SDS-PAGE under nonreduced or reduced conditions. Nonreduced proinsulin appeared as a predominant faster migrating species and two slower migrating forms (contained within bracket area), and these forms collapsed into a single band as noted under reduced conditions. The full-length resolving gel does not recover obvious evidence of higher molecular mass proinsulin complexes although labeled proinsulin recovery near the top of the resolving gel (10) cannot be excluded. B, 293T cells transfected either with empty vector (lanes 1 and 6) or proinsulin cDNA (lanes 2, 3, and 7) were pulse labeled as in A but without chase. After immunoprecipitation with anti-insulin, the samples were analyzed by nonreducing Tris-Tricine-urea-SDS-PAGE in comparison with a chemically purified proinsulin standard (lane 5, marked “125I”). The nonreduced gel was dried without fixation and after autoradiography, the slower migrating proinsulin forms from lane 3 were individually cut from the dried nonreducing gel, separately from the major proinsulin species (which had co-migrated with chemically purified 125I-proinsulin). Each excised band was re-swollen and boiled in fresh gel sample buffer plus 100 mM dithiothreitol, and analyzed in a second gel in which the original samples were also run under reducing conditions (lanes 6 and 7), along with a reduced, purified proinsulin standard (“125I,” lane 11). C, transfected 293T cells were pulse labeled as in A and chased for the times indicated. A small quantity of the nonnative proinsulin isomers is secreted but this secretion clearly does not go as efficiently to completion as is the case for the faster migrating native proinsulin band. D, two exposures of pulse-labeled 293T cells transfected with an empty vector (Control) or cDNAs encoding the wild-type human proinsulin (Proins.) or proinsulin bearing the point mutations indicated. The cells were pulse-labeled for 90 min and chased for 2.5 h, and both cell lysates (“C”) and chase media (“M”) were analyzed by immunoprecipitation with anti-insulin. In addition to the native proinsulin isomer that was very efficiently secreted over 2.5 h, two slower migrating forms of proinsulin were arbitrarily designated isomer 1 and isomer 2 bands, and these were secreted with decreased efficiency. The C(A11)S mutant was comprised of forms that co-migrated primarily with native proinsulin and isomer 1, while the C(A20)A mutant yielded a nonsecreted product that co-migrated with the isomer 2 band of proinsulin.
FIG. 3
FIG. 3. Additional single cysteine point mutants of proinsulin analyzed by nonreducing Tris-Tricine-urea-SDS-PAGE
293T cells were mock-transfected (Control) or transfected with a cDNA encoding wild-type human proinsulin or that contained the Akita mutation C(A7)Y (A) or C(B19)A (B). In A, the cells were radiolabeled for 1 h, in duplicate, and lysed without chase. The hAkita mutant was recovered at decreased levels and the band co-migrated largely with the proinsulin isomer 1 band. In B, the cells were radiolabeled for 90 min and chased for the times indicated, in the absence or presence of 20 μM MG115, a proteasome inhibitor. The C(B19)A mutant also exhibited a mobility consistent with nonnative proinsulin, was not secreted, and was degraded over 5 h in a manner partially protected by the proteasome inhibitor.
FIG. 4
FIG. 4. Newly synthesized proinsulin in the isolated pancreatic islets from wild-type and PERK−/− mice
Fifty islets were analyzed in each sample. All islets were preincubated for 40 min at the respective glucose concentrations of either 5.5 or 17 mM. The islets were then radiolabeled for 20 min with 35S-labeled amino acid mixture in the same medium. The islets were then lysed and immunoprecipitated with anti-insulin, and analyzed by nonreducing Tris-Tricine-urea-SDS-PAGE (shown in upper panel). The supernatant of the insulin immunoprecipitations was then analyzed by conventional Laemmli SDS-PAGE (shown in the lower panel). (As a standard in the Tris-Tricine-urea-SDS-PAGE above, beta-TC3 cells were pulse-labeled for 30 min with 35S-labeled amino acid mixture and chased for 30 min before cell lysis and insulin immunoprecipitation.) Note that the islets of PERK−/− mice hypersynthesize native proinsulin as well as the slower migrating species of proinsulin (downward arrows) that do not co-migrate with proinsulin conversion intermediates (Conv. Int.) (bracket).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Huang XF, Arvan P. J Biol Chem. 1995;270:20417–20423. - PubMed
    1. Guo ZY, Shen L, Feng YM. Biochemistry. 2002;41:1556–1567. - PubMed
    1. Guo ZY, Shen L, Feng YM. Biochemistry. 2002;41:10585–10592. - PubMed
    1. Zhang B-y, Liu M, Arvan P. J Biol Chem. 2003;278:3687–3693. - PubMed
    1. Hua QX, Chu YC, Jia W, Phillips NF, Wang RY, Katsoyannis PG, Weiss MA. J Biol Chem. 2002;277:43443–43453. - PubMed

Publication types

MeSH terms

LinkOut - more resources