The ER Chaperones BiP and Grp94 Regulate the Formation of Insulin-Like Growth Factor 2 (IGF2) Oligomers

Abstract

While cytosolic Hsp70 and Hsp90 chaperones have been extensively studied, less is known about how the ER Hsp70 and Hsp90 paralogs (BiP and Grp94) recognize clients and influence their folding. Here, we examine how BiP and Grp94 influence the folding of insulin-like growth factor 2 (IGF2). Full-length proIGF2 is composed of an insulin-like hormone and an E-peptide that has sequence characteristics of an intrinsically disordered region. We find that the E-peptide region allows proIGF2 to form oligomers. BiP and Grp94 influence both the folding and the oligomerization of proIGF2. BiP and Grp94 exert a similar holdase function on proIGF2 folding by preferentially binding the proIGF2 unfolded state, rather than stabilizing specific folding intermediates and changing the proIGF2 folding process. In contrast, BiP and Grp94 exert counteracting effects on proIGF2 oligomerization. BiP suppresses proIGF2 oligomerization under both ADP and ATP conditions. Interestingly, Grp94 can enhance proIGF2 oligomerization whenGrp94 adopts an open conformation (ADP conditions), but not when Grp94 is in the closed conformation (ATP conditions). We propose that BiP and Grp94 regulate the assembly of proIGF2 oligomers, and that regulated oligomerization may enable proIGF2 to be effectively packaged for export from the ER to the Golgi.

Electrostatics cause the molecular chaperone BiP to preferentially bind oligomerized states of a client protein

“…Because the hydrodynamic radius (R H ) of proIGF2 and E-peptide oligomers are in the range of hundreds of nanometers (Supplemental Figure 1A), much larger than BiP (R H ∼ 3nm 10 ), we reasoned that the binding of BiP to these oligomers could be measured by fluorescence depolarization (FP). Specifically, if BiP preferentially binds monomers or small oligomers then a negligible increase in polarization is expected, due to the small size of proIGF2 (17 kDa) relative to BiP (70 kDa), whereas if BiP preferentially binds large oligomers then a large change in polarization is anticipated (Figure 2A).…”

“…Following the signal sequence is the 67 residue mature hormone region and 89 residue positively-charged E-peptide (Figure 1B). Folded, a-helical mature IGF2 contains three disulfide bonds, whereas the E-peptide is predicted to be mostly disordered and has minimal secondary structure 10 . Once folded, proIGF2 is translocated from the ER to the Golgi for further processing 11 .…”

“…Previous work demonstrated that proIGF2 forms dynamic oligomers, where the E-peptide region is necessary for oligomerization 10 . BiP and the ER Hsp90 paralog Grp94 regulate the assembly of these oligomers while exerting only a minimal influence on the folding of proIGF2 10 . It was left unknown where and how BiP and Grp94 interact with proIGF2.…”

The endocrine and epigenetic impact of persistent cow milk consumption on prostate carcinogenesis

“…Bovine and human MEX and their miRs resist degradative conditions in the gastrointestinal tract, reach the systemic circulation, and distribute in various tissues [420,[427][428][429][430][431][432][433][434] . In fact, increasing evidence presented by studies in humans and animal models supports the view that MEX and their miRs are bioavailable, reach the systemic circulation [420,422,[434][435][436][437] , and modify gene expression of the milk recipient [359,418,[437][438][439] . It has been demonstrated that bovine MEX increased the expression of GRP94 [253] , which is a key endoplasmic reticulum chaperone enhancing the synthesis of insulin, IGF-1, and IGF-2 [254,255,440] .…”

“…Milk-derived bovine miR-148a is thus able to affect gene expression of human milk consumers (crossspecies communication) [473] . DNMT1 is a major target of miR-148a [109,110] , which explains MEX-mediated suppression of DNMT1 expression [418,438] , a pivotal postnatal mechanism modifying epigenetic regulation activating mTORC1 signaling [153,439,443,474] . In fact, DNMT1 inhibition upregulates the expression of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) [475] , which promotes the expression of mTOR (MTOR) [476] .…”