Evolution of insulin at the edge of foldability and its medical implications

Abstract

Proteins have evolved to be foldable, and yet determinants of foldability may be inapparent once the native state is reached. Insight has emerged from studies of diseases of protein misfolding, exemplified by monogenic diabetes mellitus due to mutations in proinsulin leading to endoplasmic reticulum stress and β-cell death. Cellular foldability of human proinsulin requires an invariant Phe within a conserved crevice at the receptor-binding surface (position B24). Any substitution, even related aromatic residue TyrB24, impairs insulin biosynthesis and secretion. As a seeming paradox, a monomeric TyrB24 insulin analog exhibits a native-like structure in solution with only a modest decrement in stability. Packing of TyrB24 is similar to that of PheB24, adjoining core cystine B19–A20 to seal the core; the analog also exhibits native self-assembly. Although affinity for the insulin receptor is decreased ∼20-fold, biological activities in cells and rats were within the range of natural variation. Together, our findings suggest that the invariance of PheB24 among vertebrate insulins and insulin-like growth factors reflects an essential role in enabling efficient protein folding, trafficking, and secretion, a function that is inapparent in native structures. In particular, we envision that the para-hydroxyl group of TyrB24 hinders pairing of cystine B19–A20 in an obligatory on-pathway folding intermediate. The absence of genetic variation at B24 and other conserved sites near this disulfide bridge—excluded due to β-cell dysfunction—suggests that insulin has evolved to the edge of foldability. Nonrobustness of a protein’s fitness landscape underlies both a rare monogenic syndrome and “diabesity” as a pandemic disease of civilization.

Distinct states of proinsulin misfolding in MIDY

“…Moreover, natural self-association properties of proinsulin enable the defective MIDY gene product to associate with and dominantly block export of WT proinsulin [10,11,34]. Nevertheless, different MIDY mutations lead to different ages of diabetes onset, suggesting greater or lesser severity of proinsulin misfolding [4,35]. In this study, we've examined seven MIDY proinsulin mutants by testing for the importance of Cys(A6) and Cys(A11) because, curiously, these residues normally engage in an intramolecular disulfide bond that is nonessential for proinsulin trafficking [18,19] yet in MIDY they may participate in intermolecular disulfide bonds that could render mutant proinsulin molecules unacceptable to ER quality control.…”

“…Transfected INS832/13 were incubated in media lacking Cys and Met for 30 min; 35 S-pulse-labeled for 10-30 min at 37 °C (Tran 35 S label, Perkin-Elmer) and chased in complete growth media; at chase time, media were collected. Before lysis, cells were washed with ice-cold PBS containing 20 mM N-ethylmaleimide (NEM), and then lysed in RIPA buffer (25 mM Tris, pH 7.5, 100 nM NaCl, 1% Triton X-100, 0.2% deoxycholic acid, 0.1% SDS, 10 mM EDTA) [or, in Fig.…”

“…c, d INS832/13 cells were transfected to express myctagged human lose-A6/A11 proinsulin or that bearing MIDY mutants as indicated. At 48 h post-transfection, the cells were pulse-labeled with 35 S-labeled amino acids for 30 min. c Samples were lysed immediately, processed as in Fig.…”

Distinct states of proinsulin misfolding in MIDY

“…Transfected INS832/13 were incubated in media lacking Cys and Met for 30 min; 35 S-pulse-labeled for 10 -30 min at 37°C (Tran 35 S label, Perkin Elmer) and chased in complete growth media; at chase time, media were collected. Before lysis, cells were washed with ice-cold PBS containing 20 mM N-ethyl maleimide (NEM), and then lysed in RIPA buffer (25 mM Tris, pH 7.5, 100 nM NaCl, 1% Triton X-100, 0.2% deoxycholic acid, 0.1% SDS, 10 mM EDTA) [or, in Fig.…”

Predisposition to Proinsulin Misfolding as a Genetic Risk to Diet-Induced Diabetes

“…Proinsulin exhibits significant sequence variation throughout evolution, including within both the C-peptide and insulin moieties (14; 15). The Ins gene product continues to be under selection pressure both for the ultimate bioactivity (of insulin) and for the ability of proinsulin to be folded for export through the secretory pathway of pancreatic β-cells (16). Recent evidence suggests that, with only natural variation provided by evolution, "WT" proinsulin itself is capable of forming nonnative disulfide-linked proinsulin complexes not unlike those triggered by MIDY proinsulin mutations (17).…”