Dominant protein interactions that influence the pathogenesis of conformational diseases
Jordan Wright, … , Ming Liu, Peter Arvan
Jordan Wright, … , Ming Liu, Peter Arvan
Published June 3, 2013
Citation Information: J Clin Invest. 2013;123(7):3124-3134. https://doi.org/10.1172/JCI67260.
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Research Article Endocrinology

Dominant protein interactions that influence the pathogenesis of conformational diseases

Abstract

Misfolding of exportable proteins can trigger endocrinopathies. For example, misfolding of insulin can result in autosomal dominant mutant INS gene–induced diabetes of youth, and misfolding of thyroglobulin can result in autosomal recessive congenital hypothyroidism with deficient thyroglobulin. Both proinsulin and thyroglobulin normally form homodimers; the mutant versions of both proteins misfold in the ER, triggering ER stress, and, in both cases, heterozygosity creates potential for cross-dimerization between mutant and WT gene products. Here, we investigated these two ER-retained mutant secretory proteins and the selectivity of their interactions with their respective WT counterparts. In both cases and in animal models of these diseases, we found that conditions favoring an increased stoichiometry of mutant gene product dominantly inhibited export of the WT partner, while increased relative level of the WT gene product helped to rescue secretion of the mutant partner. Surprisingly, the bidirectional consequences of secretory blockade and rescue occur simultaneously in the same cells. Thus, in the context of heterozygosity, expression level and stability of WT subunits may be a critical factor influencing the effect of protein misfolding on clinical phenotype. These results offer new insight into dominant as well as recessive inheritance of conformational diseases and offer opportunities for the development of new therapies.

Authors

Jordan Wright, Xiaofan Wang, Leena Haataja, Aaron P. Kellogg, Jaemin Lee, Ming Liu, Peter Arvan

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Figure 1

Proinsulin-KDEL interacts with and inhibits secretion of WT proinsulin.

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Proinsulin-KDEL interacts with and inhibits secretion of WT proinsulin. ...
293T cells transiently transfected with hPro-CpepMyc were cotransfected with plasmids as indicated. (A) Cell lysates (C) and media (M) were resolved by SDS-PAGE, electrotransfer, and immunoblotting (WB) with anti-Myc. The media/cell ratio of hPro-CpepMyc bands was decreased by 58.9% ± 12.8% (P = 0.003, n = 6) in cells coexpressing hPro-CpepSfGFP-KDEL compared with that in cells coexpressing WT hPro-CpepSfGFP. (B) Cells lysed in TX-CoIP buffer were immunoprecipitated with anti-GFP or anti-Myc and resolved by SDS-PAGE, electrotransfer, and immunoblotting with anti-GFP or anti-Myc as indicated. The top 2 rows demonstrate expression of the indicated proteins, and the bottom row demonstrates CoIP. Gels are representative of 3 independent experiments. (C) Cells transiently expressing WT hPro plus TgGFP were cotransfected with plasmids as indicated. The media collected overnight were analyzed by hPro-specific RIA. TgGFP in the same cell lysates and media was analyzed by SDS-PAGE, electrotransfer, and immunoblotting with anti-GFP. The media/cell ratio of TgGFP bands in cells coexpressing mPro-KDEL exhibited no significant change compared with that from cells coexpressing WT mPro (1.9 ± 0.2 vs. 2.1 ± 0.8; P = 0.3, n = 5). In B and C, noncontiguous lanes from the same gel are shown. (D) Cells transiently expressing hPro-CpepMyc were cotransfected with plasmids expressing mPro-KDEL or mPro. Media were collected overnight, and cell lysates were analyzed by hPro-specific RIA. The data in C and D represent mean ± SEM, each from ≥4 independent transfections. *P < 0.05.