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. 2020 Nov 5;117(47):29618–29628. doi: 10.1073/pnas.2010908117

Fig. 4.

Fig. 4.

Cellular folding and secretion properties of proinsulin variants in HEK293T cells. (A) Assessment of trans-dominance of B24 proinsulin variants in HEK293T cells. Gel depicts 35S-labeled proteins in cell lysates at the end of 30-min 35S-labeling (leftmost lanes marked “C”) and after 90-min chase (middle lanes marked “C”). Samples collected from chase media were run in lanes marked “M.” Bands correspond to Myc-tagged WT proinsulin and untagged proproteins are as labeled at right. Lanes marked “Con” (control) indicate nontransfected cells. Bands corresponding to myc-tagged proinsulin are visible in the media of cells cotransfected with WT-untagged proinsulin and, more faintly, on coexpression of SerB24, GlyB24, MetB24, and LeuB24 proinsulins. In contrast, the corresponding band is markedly attenuated (or not detectable) in media from cells cotransfected with CysB24 or TyrB24 variants. Portions of panel reprinted from ref. 35 with permission of the authors and included here for clarity and convenience. (B) Metabolic labeling of newly synthesized WT and variant preproinsulin (pPI) and proinsulin (PI) analyzed by Tris-tricine-urea SDS/PAGE under nonreducing (Upper) and reducing (Lower) conditions; bands in nonreducing SDS/PAGE represent natively folded proinsulin or nonnative isomers. (C) Secretability of WT or variant proinsulins in HEK293T cells. Differences from WT were in each statistically significant (P < 0.05). (D) Cartoon depicting BiP activation in unfolded-protein response (UPR). Accumulation of unfolded proteins (red coil) within the ER leads to the dissociation of BiP (blue semicircle) from ATF6 (brown), IRE-1 (purple), and PERK (orange). Liberated BiP then binds to an unfolded protein to prevent proteotoxic aggregation. Activated ATF6, IRE-1, and PERK initiate UPR, decreasing translation of most proteins within the cell and increasing ER-associated degradation (ERAD). Expression of ER-associated chaperones, including BiP, is up-regulated by UPR. (E) UPR activation by WT or variant proinsulins as monitored by a BiP-luciferase reporter in MIN6 cells; *P < 0.05 relative to WT baseline, whereas #P < 0.05 relative to the elevated level of ER stress induced by CysB24 proinsulin.