If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Division of Metabolism, Endocrinology and Diabetes, University of Michigan Medical Center, Ann Arbor, Michigan 48105, AustraliaDepartment of Endocrinology and Metabolism, Tianjin Medical University General Hospital, Tianjin, Heping District, 300052 China
1 Predoctoral Fellow of the National Institutes of Health, Medical Scientist Training Program, supported by Core Grant 5T32GM007250-38 and Fellowship 1F30DK112644. 2 Supported in part by the American Diabetes Association Grants 7-13-IN-31 and 1-08-RA-149.
Globular protein sequences encode not only functional structures (the native state) but also protein foldability, i.e. a conformational search that is both efficient and robustly minimizes misfolding. Studies of mutations associated with toxic misfolding have yielded insights into molecular determinants of protein foldability. Of particular interest are residues that are conserved yet dispensable in the native state. Here, we exploited the mutant proinsulin syndrome (a major cause of permanent neonatal-onset diabetes mellitus) to investigate whether toxic misfolding poses an evolutionary constraint. Our experiments focused on an invariant aromatic motif (PheB24–PheB25–TyrB26) with complementary roles in native self-assembly and receptor binding. A novel class of mutations provided evidence that insulin can bind to the insulin receptor (IR) in two different modes, distinguished by a “register shift” in this motif, as visualized by molecular dynamics (MD) simulations. Register-shift variants are active but defective in cellular foldability and exquisitely susceptible to fibrillation in vitro. Indeed, expression of the corresponding proinsulin variant induced endoplasmic reticulum stress, a general feature of the mutant proinsulin syndrome. Although not present among vertebrate insulin and insulin-like sequences, a prototypical variant ([GlyB24]insulin) was as potent as WT insulin in a rat model of diabetes. Although in MD simulations the shifted register of receptor engagement is compatible with the structure and allosteric reorganization of the IR-signaling complex, our results suggest that this binding mode is associated with toxic misfolding and so is disallowed in evolution. The implicit threat of proteotoxicity limits sequence variation among vertebrate insulins and insulin-like growth factors.
The structure and function of globular proteins are determined by their sequences. Yet the informational content of such sequences must also contain determinants of folding efficiency, providing safeguards against toxic misfolding (
). In this study, we demonstrate that insulin can in principle exhibit two modes of receptor binding, but only one protects from toxic misfolding. Evidence is provided that the excluded mode would enhance the risk of pancreatic β-cell dysfunction and diabetes mellitus (DM)
Of particular interest are DM-associated mutations in proinsulin not involving cysteine. Whereas patients with Cys-related mutations invariably present with neonatal DM, nonCys-related mutations are associated with ages of onset ranging from neonatal to the 3rd decade of life; furthermore, the latter pedigrees typically exhibit incomplete penetrance (
). In the classical structure of insulin, this turn enables the C-terminal B chain β-strand (residues B24–B28) to pack against the conserved side chains of IleA2, ValA3, LeuB11, ValB12, and LeuB15. These contacts seal the hydrophobic core of the α-helical domain. On insulin self-assembly, the β-strand forms a dimer-related antiparallel β-sheet (Fig. S2) (
). On receptor binding, a conformational change ensues: rotation of the B20–B23 β-turn and adjoining PheB24–PheB25 element leads to detachment of the C-terminal B chain β-strand from the core. The β-strand itself packs in a groove between IR-ectodomain elements L1 and αCT (Fig. S3A), wherein the side chain of PheB24 packs within a nonpolar pocket; the side chains of PheB25 and TyrB26 occupy more peripheral sites (Fig. S3, A and B) (
Substitutions at B24–B26 uncovered distinct and specific side-chain determinants of IR binding. The B24-binding pocket (defined by residues in L1, αCT, and the central B chain α-helix) optimally accepts Phe but can also accommodate cyclohexylalanine (Cha), Met, or branched aliphatic side chain; Tyr, His, and Trp are disfavored despite their aromaticity (
), substitution of PheB24 by Gly would seem to leave a destabilizing cavity (blue box in Fig. 1E). How might native affinity and activity be regained? A potential framework is provided by the register-shift model; an alternative receptor-binding mode of insulin in which residue B24 reorients as part of a noncanonical five-residue chain reversal (B20–B24), in turn enabling residues B25–B27 to engage the respective B24-, B25-, and B26-binding pockets of the IR (
). Such a shift would (i) preserve insertion of a Phe within the B24-binding pocket (PheB25), (ii) deploy a well-tolerated Tyr within the B25-binding pocket (TyrB26), and (iii) exploit the promiscuity of the solvated B26-binding surface to accommodate a small polar side chain (ThrB27) (illustrated in Fig. 1F).
This study provides mutational evidence in support of the register-shift model and suggests that this alternative mode of binding has been excluded in the evolutionary history of the vertebrate insulin family by toxic protein misfolding. Our results are discussed in relation to molecular dynamics (MD) simulations of [GlyB24]insulin/IR ectodomain complexes based on the crystal structure of insulin bound to its primary IR-binding site (PDB entry 4OGA) (
). Because of the chemical similarities between Lys and Orn, [OrnB29]insulin exhibits native-like biological and biophysical properties. In accordance with the classical studies of Tager and co-workers (
), substitution PheB24 → Gly preserved IR-binding affinity in a competitive displacement assay (Table 1 and Fig. S4A). SerB24 by contrast impaired binding by ∼25-fold. Although similar qualitative trends in biological activity were observed (as assessed in rats rendered diabetic by streptozotocin (STZ) (
No classical pocket exists at the B25-cognate surface of the receptor. Binding is markedly impaired by a tetrahedral γ-carbon in the B25 side chain (such as in Leu or Cha), highlighting a geometric requirement for trigonal sp2 hybridization at Cγ (
Given these distinct site-specific SARs, the register-shift mode would lead to successive SAR shifts (Fig. 1, D–F). Whereas a tetrahedral γ-carbon in the B25 side chain (Leu or Cha) would ordinarily impede the binding of insulin, for example, the same modification in the context of a GlyB24 analog might be “rescued” as the variant B25 side chain could then be well-accommodated within the B24-binding pocket. Similarly, whereas SerB26 ordinarily confers high activity, in a shifted complex SerB26 would be directed onto the incompatible B25-binding surface (Fig. S5).
Comparative studies of paired insulin analogs provide critical tests of model
To test such predictions, a series of paired PheB24/GlyB24 analogs were prepared. Probes of respective B24–B26-binding sites were provided by ChaB25, LeuB25, TyrB25, and SerB26 (Fig. S5). The pattern of receptor-binding affinities was found to be in broad agreement with the predictions (Fig. 2 and Table 2, column 3). Particularly striking is the rescue by GlyB24 of otherwise unfavorable B25 substitutions as exemplified by the tetrahedral γ-carbons of LeuB25 and ChaB25. Conversely, introduction of GlyB24 rendered TyrB25 and SerB26 (ordinarily well-tolerated) unfavorable. Whereas this pattern of affinities would seem paradoxical in the context of the native hormone/receptor complex (
To assess the in vivo relevance of these receptor-binding studies, biological activities were tested in STZ rats (Fig. 3 and Table 2, column 2). Pharmacodynamic (PD) results were broadly in accordance with the in vitro data, including GlyB24 rescue of minimally-active [LeuB25]insulin and [ChaB25]insulin analogs. Indeed, the double mutants exhibited activities similar to those of the corresponding B24 analogs. Conversely, addition of GlyB24 to super-active TyrB25 or SerB26 analogs led to impaired function, similar to respective TyrB24 or SerB25 analogs. Relative biological potencies were estimated based on areas under the PD curves (AUCs) as given in Table 2.
Systematic characterization of the complete set of analogs (15 in total) was obtained in cell-based assays. The assays employed human breast cancer cell line MCF-7 (
), demonstrated that GlyB24 attenuates stability (ΔΔGu 1.3(±0.2) kcal/mol relative to parent [OrnB29]insulin (Fig. 5, A and B, and Table 3)). SerB24 and LeuB24 were likewise destabilizing (ΔΔGu 1.3(±0.2) and 1.3(±0.2) kcal/mol, respectively; positive values represent loss of stability in the difference ΔGu[mutant] − ΔGu[parent], where the parent analog is [OrnB29]insulin). These thermodynamic decrements are in accordance with the structural role of PheB24 at the edge of the hydrophobic core (
), its substitution by LeuB25 or ChaB25 was also observed to impair thermodynamic stability (although to a lesser extent than the B24 substitutions): ΔΔGu 0.7(±0.2) and 0.9(±0.2) kcal/mol, respectively. Such destabilization may in part reflect a solvation free-energy penalty (
). In contrast to the above functional rescue, co-introduction of GlyB24 accentuated the destabilizing effects of LeuB25 and ChaB25. The combination of GlyB24 and LeuB25 impaired stability (ΔΔGu 1.9(±0.2) kcal/mol), whereas the combination of GlyB24 and ChaB25 kcal/mol impaired stability by 1.5(±0.2) kcal/mol (Fig. S8 and Table S1). These additive perturbations are uncorrelated with effects on activity.
PheB24 stabilizes native self-assembly and metal-ion coordination
Protein self-association (in the absence of zinc ions or phenolic ligands) was probed by size-exclusion chromatography (SEC) (
). A range of elution times and peak shapes was observed in relation to WT insulin (earliest eluting) and engineered monomer (KP-insulin; latest eluting) (Fig. 5C); molecular masses were inferred in reference to these standards (Fig. S9 and Table 4). Whereas WT insulin and [OrnB29]insulin were essentially dimeric under these conditions (inferred molecular masses 6.8 and 6.6 kDa, respectively), the SerB24 and GlyB24 analogs exhibited elution profiles intermediate between monomer and dimer (molecular masses in each case 4.9 kDa). Such variant dimers are presumably destabilized by a cavity at the dimer interface, comprising the contiguous volumes occupied by PheB24 and its dimer-related mate.
Table 4Self-association properties of insulin analogs
). The hexamer lifetime of [GlyB24,OrnB29]insulin (t½ = 1.2(±0.2) min) was profoundly reduced relative to [OrnB29]insulin (8.2(±0.8) min), WT insulin (8.2(±1.3) min), or even the rapidly-dissociating analog insulin lispro
(KP) (4.6(±0.3) min). The hexamer lifetime of [SerB24,OrnB29]insulin was also reduced, but ∼2-fold greater than that of [GlyB24,OrnB29]insulin. PheB24 therefore contributes to a kinetic barrier that locks the R6 insulin hexamer (Fig. 5, D and E.)
A qualitative probe for the dynamic stability of the tetrahedral Co2+-coordination site within the R6 hexamer is provided by the magnitude of the 574 nm d–d absorbance band; this spectral feature would be absent in an octahedral site. The Co2+ d–d band was attenuated in the GlyB24 analog relative to the other proteins, including [SerB24,OrnB29]insulin and insulin lispro (Fig. 5, D and E, and Table 4). These data suggest that the GlyB24-analog hexamer contains a distorted coordination site and/or that this site exhibits local dynamic interconversion between tetrahedral and octahedral structures. Either mechanism would represent a transmitted perturbation as the mutation site (B24) is distant from the HisB10-related site of metal-ion coordination (
PheB24 protects insulin from fibrillation, a toxic misfolding process
Susceptibilities of the GlyB24 and SerB24 analogs to the formation of insoluble amyloid fibrils (Fig. 5F) were evaluated (in the absence of metal ions) relative to WT insulin, [OrnB29]insulin, and insulin lispro. Aliquots were agitated at a protein concentration of 60 μm at 37 °C. Lag times were measured using thioflavin T (ThT) as a fluorescent probe of cross-β assembly (
). The B24 analogs exhibited shorter lag times than the control samples (Fig. 5G and Table 5). Comparison of corresponding single and double mutants demonstrated that GlyB24 also accelerated the fibrillation of analogs containing substitutions at positions B25 or B26 (Fig. S11 and Table S2; data obtained at room temperature using “gentle sloshing” in glass vials). We envision that, under native conditions, the B24 substitutions enhance subpopulations of amyloidogenic partial folds as components of a conformational equilibrium (
Fibrillation lag times pertain to zinc-free WT insulin and analogs (in a monomer–dimer equilibrium); each protein was made 60 μm in phosphate-buffered saline (pH 7.4). Samples were agitated via continuous shaking at 37 °C. Time of initial fluorescence, defined as a 2-fold increase over baseline in ThT fluorescence, provided a criterion for onset of fibrillation.
h ± S.D. (n)
8.3 ± 3.4 (8)
14.0 ± 1.4 (7)
3.3 ± 0.3 (7)
2.7 ± 0.3 (6)
6.2 ± 2.8 (7)
a Fibrillation lag times pertain to zinc-free WT insulin and analogs (in a monomer–dimer equilibrium); each protein was made 60 μm in phosphate-buffered saline (pH 7.4). Samples were agitated via continuous shaking at 37 °C. Time of initial fluorescence, defined as a 2-fold increase over baseline in ThT fluorescence, provided a criterion for onset of fibrillation.
). Nascent proteins were pulse-labeled with [35S]cysteine/methionine for 30 min followed by a 2-h chase. Oxidative folding and secretion of [GlyB24]proinsulin were evaluated using nonreducing Tris–Tricine–urea–SDS-PAGE (Fig. 6A, the c and m refer to the cells and chase media, respectively) (
). [GlyB24]Proinsulin exhibited impaired folding efficiency (relative to WT) as demonstrated by a weaker band corresponding to native proinsulin and greater relative prominence of bands corresponding to non-native disulfide isomers. In accordance with previous studies (
), SerB24 led to similar qualitative perturbations, but with less marked attenuation of the native band in the cellular fraction (Fig. S13).
Additional experiments were performed to assess protein trafficking and secretion. The variant proinsulins exhibited preferential secretion of non-native disulfide isomers (upper bands in lanes “m” in Fig. 6A and Fig. S13). Radiolabeled WT or variant proinsulin in cell lysates and post-chase media was quantified; relative secretion efficiencies of [GlyB24]proinsulin and [SerB24]proinsulin were 25 and 40%, respectively (Fig. 6B). Further insight was obtained through fluorescence studies of variant proinsulins containing green fluorescent protein (GFP) within the C domain, as described previously (
). This chimeric construct is shown in schematic form in Fig. S14.
In rat INS1 β-cells, WT-proinsulin–GFP chimera exited from the ER were stored in the secretory granules and exhibited a punctate secretory–granule pattern that did not co-localize with an ER marker (Fig. 6C, red). Although previously, the [CysB24]proinsulin–GFP (a mutation associated with neonatal-onset DM) was severely misfolded and completely retained in the ER (Fig. S15), SerB24 and GlyB24 were partially exported from the ER (Fig. 6C). It is possible that partial ER exit of the B24 variants in these assays was due to co-expression of WT rat proinsulins, which might act in trans to rescue trafficking of the human chimeric variants (
), we next exploited a resident ER quality-control sensor (binding immunoglobulin protein; BiP), constructed as a luciferase fusion protein, to probe the extent of proinsulin misfolding (Fig. S16). Luciferase activity was stimulated in the order WT < GlyB24, SerB24 < CysB24 (Fig. 6D). In addition to its the structural roles highlighted above (i.e. receptor binding, native-state stability, self-assembly, and protection from fibrillation), PheB24 thus contributes to the folding efficiency of proinsulin in the ER and its secretability.
Together, these cell-based studies suggest that potential inheritance of GlyB24 in a vertebrate would enhance the risk of β-cell dysfunction, rationalizing its evolutionary exclusion despite the native biological function of the mature hormone. Comparison with MIDY mutations CysB24 (neonatal) and SerB24 (onset in early adulthood) suggests that in humans a GlyB24 variant INS gene would lead to DM onset in adolescence.
The Anfinsen paradigm posits that the sequence of a protein determines its structure and stability (
), highlighting the complementary requirement for folding efficiency, has long stimulated investigation into biophysical mechanisms by which a folding chain avoids the need for an exhaustive conformational search (
). This study was motivated by the notion that the informational context of protein sequences can either be overt (apparent in the native structure), implicit (critical to folding efficiency but dispensable in the native state, once obtained), or cryptic (safeguards against off-pathway events) (
). To this end, this study has focused on an invariant aromatic residue (PheB24) that makes critical contributions to structure, assembly, and function. A site of clinical mutations associated with DM (
), position B24, illustrates the remarkable compression of information that is possible within the sequence of a globular protein. The rigorous conservation of PheB24 in vertebrates presumably reflects the intersection of multiple distinct constraints, from biosynthesis to function, that have governed the evolution of insulin-related sequences for >500 million years (Fig. S1).
Anomalous structure–activity relationships
A starting point for this study was provided by a long-standing enigma: the native activity of [GlyB24]insulin. Originally regarded as anomalous by Tager and co-workers (
). Detachment of the B24–B28 segment from the α-helical core of insulin was proposed both to liberate the “aromatic triplet” (PheB24–PheB25–TyrB26) as a conserved receptor-binding motif and to expose part of the underlying core to enable its further receptor engagement (
). Indeed, the aromatic triplet was observed to pack within a groove between ectodomain elements L1 and αCT; their displacement indeed enables receptor engagement by conserved nonpolar side chains in the central B chain α-helix and N-terminal A chain α-helix (
). In addition, recent structures of insulin bound to the intact IR ectodomain (obtained by cryo-electron microscopic (cryo-EM) single-particle image reconstruction) have revealed that binding of the detached B24–B28 β-strand is coupled to large-scale reorganization of domains within the dimeric assembly, a plausible first step in transmembrane signal propagation (
In the hormone/ectodomain complex, the side chain of PheB24 inserts within a classical nonpolar pocket, with boundaries formed by aromatic and aliphatic side chains in L1, αCT, and the central B chain α-helix (
), posits that the C-terminal segment of the insulin B chain may undergo a shift in alignment relative to the B24-, B25-, and B26-binding sites in the receptor. In this model, GlyB24 or d-amino acid substituents would not themselves engage the receptor; rather, their destabilizing effects would enable “slippage” of the B chain such that residues B25–B27 would align with the B24–B26 receptor sites. The side chain of PheB25 would thus replace PheB24 in the B24-binding pocket; TyrB26 would replace PheB25 at an adjoining surface of αCT, and ThrB27 would replace TyrB26 at a peripheral surface near the L1–αCT interface. The latter, although a seemingly nonconservative substitution, would be in accordance with the intrinsic compatibility of the B26-binding surface of the ectodomain for small polar side chains (
The register-shift model made specific yet counter-intuitive predictions that GlyB24 could rescue the function of otherwise unfavorable substitutions at B25 or B26 and, conversely, render unfavorable substitutions at these positions otherwise compatible with high activity. Thus, whereas a tetrahedral γ-carbon (such as in the aliphatic side chains of Leu or Cha) is ordinarily excluded at residue B25, such side chains could readily dock within the B24-related pocket via a register shift and hence be rescued as a mechanism-based example of second-site compensation. Conversely, Phe and Tyr are each well-accommodated at the B25-binding site (and indeed IGFs contain Tyr at this position (
)). Together, these results are in accordance with such reasoning and so provide strong evidence in support of the register-shift hypothesis.
Systematic screening of analog activities in human breast cancer cell line MCF-7 (assessinginsulin-dependent IR autophosphorylation and downstream transcriptional regulation) provided additional evidence in support of the register-shift model. This complete set of activities (15 analogs in total) corroborated selected measurements of receptor-binding affinities and in vivo potencies. Together, these results suggest that the native and register-shift modes of insulin binding lead to similar structural reorganization of the IR ectodomain and mechanisms of transmembrane signal propagation (
To explore potential structural mechanisms underlying the proposed register shift, two models of the interface between [GlyB24]insulin and the IR ectodomain were constructed. Modeling was based on the crystal structure of WT insulin in complex with the primary IR binding site (PDB code 4OGA) (
). Relative to the WT complex (Fig. 7A), a naïve model of the [GlyB24]insulin complex was first constructed without a shift, i.e. PheB24 was substituted by Gly in the context of the native structure (Fig. 7B). In this model, such a binding mode would leave a cavity in the space ordinarily occupied by the B24 aromatic ring with an average volume of 27.3(±0.5) Å3 across the simulation. Such a large cavity would presumably incur a free-energy penalty (
)) would be 2.12 kcal/mol. Although such a model illustrates how the WT complex could accommodate a large–to–small substitution without transmitted conformational changes, it appears inconsistent with the present functional studies of insulin analogs.
To allow for structural rearrangements near the site of substitution, the modeling procedure next exploited the unoccupied space in the WT structure near the native B20–B23 β-turn. This space would readily accommodate a five-residue noncanonical turn (Gly–Glu–Arg–Gly–Gly) without steric clash such that the side chain of PheB25 can orient toward and partially fill the B24-binding pocket (Fig. 7C). The root mean square deviation between this model and the WT complex (after MD simulation) is 1.9 Å. In this model TyrB26 inserts into the B25-binding cleft (between αCT residues Val715 and Pro718), thereby mimicking the role of PheB25 in WT complexes. MD simulations of all three binding modes (WT insulin in the native complex (Fig. 7D), [GlyB24]insulin in the naïve model (Fig. 7E), and [GlyB24]insulin in a shifted model (Fig. 7F)) indicated that such modes were stable throughout simulations. Whereas in the unshifted mode only the main-chain atoms of GlyB24 can mitigate the otherwise empty B24-binding pocket, in the register-shifted model this potential space is more efficiently filled by the side chain of PheB25(pink spheres in Fig. 7). Estimates of residual cavity volumes in the respective models predicted the register-shift mode reduces the potential B24-related cavity by 12.9(±0.4) Å3 (Fig. S17).
We note in passing that the first mode, wherein the native PheB24-binding pocket would largely be empty, native affinity might be in part rescued from the full cavity penalty (
) by a presumed reduced free-energy cost of conformational change in the more flexible variant insulin. Although this scheme would be in accordance with general biophysical principles, it would not rationalize the present pattern of second-site effects. In particular, an empty B24-binding pocket could not rationalize why GlyB24 rendered TyrB25 unfavorable or rescued the activity of ChaB25 through second-site compensation. In the future, such entropic effects could be addressed via free-energy molecular dynamics simulations. It would also be of interest to obtain a definitive structure of a [GlyB24]insulin analog bound to the IR ectodomain, such that the status of the B24-binding pocket (empty or filled by PheB25) could be directly visualized.
We emphasize that both models assume “molecular parsimony” as potential large-scale reorganization of the hormone–receptor interface (including changes in domain–domain contacts within the dimeric ectodomain) was not considered. These more complex possibilities, if realized, would underscore the essential insight that two modes of hormone binding are possible, but one is disallowed in evolution (see below).
Our rodent studies demonstrated that the register-shift mode of receptor engagement not only permits IR binding but also can mediate metabolic regulation in a vertebrate. Indeed, [GlyB24,OrnB29]insulin was as effective as its parent [OrnB29]insulin for the short-term treatment of DM. This observation was not self-evident based on receptor-binding studies, as it was a priori possible either (a) that a shifted mode of receptor engagement could have been less active in signal propagation or (b) that the impaired stability of the GlyB24 analog could have led to its premature degradation in vivo. Given the observed maintenance of biological activity in vivo, however, we were motivated to consider why GlyB24 has apparently been disallowed in the evolution of vertebrate insulins and IGFs. This question relates to possible evolutionary constraints beyond prominence of PheB24 in the structure and function of insulin's native state. A precedent for cryptic functions of insulin residues was previously provided by PheB1, shown to be critical to the folding efficiency of proinsulin in mammalian cells, but dispensable for structure and function of the mature hormone once its folding is achieved (
). GlyB24 analogs exhibited shorter lag times prior to onset of fibrillation, suggesting that the heightened presence of conformational substrates (which may constitute a small fraction of protein molecules at any one time) are amenable to non-native aggregation en route to formation of an amyloidogenic nucleus (
). The side chain of PheB24 may thus serve to reduce the stability or accessibility of such conformational excursions relative to the predominant (native) conformation. Because in general fibrillation lag times have been found not to correlate with thermodynamic stabilities (ΔGu relative to the unfolded state), this role of PheB24 presumably relates to amyloidogenic partial folds (
). These studies have shown that substitution of PheB24 by Gly impairs native self-assembly in the absence of metal ions and reduces the kinetic stability of R6 hexamers (containing Co2+ as an isomorphic replacement for Zn2+) once formed. The intrinsic structural role of PheB24 to protect the insulin monomer from non-native aggregation is thus accompanied by a second protective function in promoting protective native self-assembly.
An insulin species variant (in the South American rodent Octadon degus) forms senile amyloid plaques in pancreatic islets; these are proposed to impair β-cell viability leading to late-onset DM (
)), and substitution HisB10 → Asn, which blocks binding of zinc ions. This species variant is thus less protected from fibrillation than other vertebrate insulins. The continued survival of this species (and thus existence of the variant insulin as an extant vertebrate sequence) presumably reflects the post-reproductive onset of DM.
PheB24 as a cryptic determinant of foldability
Proinsulin, the single-chain biosynthetic precursor of insulin (
). This syndrome (MIDY) is a major cause of permanent neonatal-onset diabetes. Whereas the majority of such mutations add or remove a cysteine (thus giving rise to an odd number of reactive thiol groups in the nascent polypeptide chain), mutations have also been identified unrelated to Cys. Such mutations presumably identify sites critical to the mechanism of disulfide pairing (
). In our cell-based studies, GlyB24 was at least as perturbing as SerB24 with respect to fidelity of disulfide pairing, efficiency of folding, induction of ER stress, and extent of secretion into the medium. Prior to the discovery of insulin for the treatment of insulin-deficient DM, onset during the reproductive years (even with variable genetic penetrance) is likely to have been sufficient to reduce reproductive fitness and so purge such a sequence variation from extant insulin genes.
Previous fluorescence-imaging studies of β-cell lines expressing proinsulin variants have defined a range of potential defects in protein biosynthesis and/or subcellular trafficking. At one extreme, MIDY mutations resulting in an odd number of cysteines (including classical Akita allele CysA7 → Tyr) typically induce severe defects in nascent folding, leading to ER sequestration and ER-associated degradation (
). By contrast, a “zip code” variant compatible with native folding (HisB10 → Asp; proinsulin Providence) readily exits the ER, but altered trafficking in the Golgi apparatus leads to storage largely within a constitutive pool of secretory granules lacking prohormone convertases and the machinery of glucose-dependent exocytosis (
). Although [AspB10]insulin exhibits enhanced affinity for the IR and augmented thermodynamic stability, the patients were found to exhibit elevated levels of circulating [AspB10]proinsulin due to its aberrant constitutive secretion
Clinical mutations in proinsulin may exhibit a range of intermediate cellular phenotypes depending on the specific amino acid substitution. Fluorescence images have thus revealed partial or near-complete sequestration of the variant proinsulin within the ER with varying degrees of impaired Golgi trafficking (
). The pattern of fluorescence conferred by GlyB24 (as probed by the C domain–inserted GFP in a proinsulin chimera) would predict onset in the 2nd or 3rd decade of life should this mutation be observed in future patients.
PheB24 thus plays a role in the ER of β-cells (to enhance the foldability of nascent proinsulin) that is distinct from its subsequent roles in the post-Golgi network (to enable productive trafficking) and in glucose-regulated secretory granules (to promote native self-assembly). These aspects of biosynthesis are unrelated to the function of PheB24 in the mature hormone (as a key contact at a conserved hormone–receptor interface). The multiple roles of this invariant residue thus illustrates the remarkable compression of coding information that is possible within a protein sequence. GlyB24 honors in the breach the importance of cell-biological mechanisms and pathological processes not discernable in the three-dimensional structures of proteins, however beautiful and compelling as molecular architectures.
GlyB24 is unique among potential amino acid mutations at B24 in that this variant insulin would retain native activity should its biosynthesis be feasible. Our results nonetheless predict significant defects in the folding, trafficking, and secretion of the variant proinsulin. It would be of future interest to test this prediction in an engineered mouse. To this end, a CRISPR/Cas9-directed GlyB24 variant could define the extent of associated β-cell dysfunction and so inform which evolutionary constraints govern its exclusion among vertebrate insulin sequences. Because the mouse contains two insulin genes (four per diploid genome), such a mouse model may also require a deletion of one WT gene (per haploid genome) to mimic the gene dosage in a human heterozygous patient with the mutant proinsulin syndrome.
The complex conformational “life cycle” of the insulin molecule—from its nascent folding within pancreatic β-cells to receptor engagement at target tissues—imposes evolutionary constraints that may be overt, implicit, or cryptic. Safeguards against proteotoxic misfolding, as exemplified by the PheB24-anchored B20–B23 β-turn, can make independent contributions to the biological function of the native state (
). Multidisciplinary dissection of such a safeguard, made possible here by a collaborative team, may in the future enable a deeper understanding of the informational context of insulin sequences. Such studies may define a subset of MIDY mutations amenable to compensation by chemical chaperones as an opportunity for precision medicine beyond insulin replacement therapy (
) is a conserved functional element of vertebrate insulins and insulin-like growth factors. A broader view of the space of insulin-like sequences would encompass invertebrate as well as vertebrate homologs. The genome of Caenorhabditis elegans, for example, encodes 38 putative insulin-like proteins with divergent features relative to vertebrate insulins (
). Remarkably, despite containing multiple core and surface substitutions, each individually disallowed in vertebrate insulin, the structure of one such nematode protein closely resembles human insulin (
). Second-site compensation among the present set of GlyB24 human insulin analogs provides proof of principle that this landscape can include novel modes of hormone–receptor recognition leading to transmembrane signaling. Although excluded in vertebrate evolution evolution, these alternative mechanisms of signaling may in principle be exploited in the design of therapeutic analogs (
). The resulting 51-residue insulin analogs were purified by preparative C4 rp-HPLC (Higgins Analytical Inc., Proto 300 C4 with 10-μm particle size and dimensions 250 × 20 mm), and their purity was assessed by analytical C4 rp-HPLC (Higgins Analytical Inc., Proto 300 C4 with 5-μm particle size and dimensions 250 × 4.6 mm). Predicted molecular masses were in each case verified using an Applied Biosystems 4700 proteomics analyzer MALDI-TOF.
Circular dichroism spectroscopy
Far-UV spectra were obtained from 200 to 250 nm on an AVIV spectropolarimeter equipped with an automated syringe-driven titration unit (
). In brief, data were fit by nonlinear least squares to a two-state-model as shown in Equation 1,
where x is the concentration of guanidine hydrochloride, and A and B represent respective estimates of the baseline ellipticities of the protein in its unfolded and native states as extrapolated to zero guanidine concentration (
). The assay employed detergent-solubilized holoreceptor with C-terminal streptavidin-binding protein tag. The receptor was purified by sequential wheat-germ agglutinin and streptactin-affinity chromatography from detergent lysates of polyclonal stably-transfected 293PEAK cell lines expressing each receptor. To obtain analog dissociation constants, competitive binding data were analyzed by nonlinear regression by the method of Wang (
Male Lewis rats (mean body mass ∼300 g) were rendered diabetic by STZ. To test potencies, the analogs were made 10 μg per 100 μl in a formulation buffer (16 mg/ml glycerin, 1.6 mg/ml meta-cresol, 0.65 mg/ml phenol, and 3.8 mg/ml sodium phosphate (pH 7.4)) and injected intravenously into tail veins (
). WT insulin or analogs were each re-purified by rp-HPLC, lyophilized, dissolved in Lilly diluent at the same maximum protein concentration, and re-quantitated by analytical C4 rp-HPLC to ensure uniformity. Dilutions were made using the above buffer.
Rats were injected at time t0. Blood was obtained from the clipped tip of the tail at time t0 and every 10 min for the 1st h, every 20 min for the 2nd h, every 30 min for the 3rd h, and every hour thereafter to a final time of 5 h. Efficacy of WT insulin or analog to reduce the blood glucose concentration was calculated using the following: (a) the rate of change in blood glucose concentration over 240 min following initial injection, and (b) the integrated area between the x axis and the curve representing fractional blood–glucose level with relation to initial blood–glucose concentration (AUC). Statistical significance was assessed using a Student's t test.
Animals used in this study were housed at the Association for Assessment and Accreditation of the Laboratory Animal Care (AAALAC)-accredited facilities of Case Western Reserve University (CWRU) School of Medicine. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) Office at CWRU, which provided Standard Operating Procedures and reference materials for animal use (in accordance with the NIH Guide for the Care and Use of Laboratory Animals). The animal health program for all laboratory animals was directed by the CWRU Animal Resource Center. Animal care and use were further monitored for Training and Compliance issues by Veterinary Services.
Signaling assays in mammalian cell culture
Signaling activities of the insulin analogs were tested in MCF-7 human breast adenocarcinoma cells (which express IR-A, IR-B, and high levels of IGF-1 receptor (
)). MCF-7 cells (American Type Culture Collection, Manassas, VA) were cultured in Eagle's minimum essential medium supplemented with 10% FBS, 1% penicillin/streptomycin, and sodium pyruvate (1 mm). 24-h serum-starving protocol using appropriate culture medium except FBS was applied at 70–75% cell confluence. After serum-starving, serum-free medium containing selected concentrations of insulin analogs was added to each well (control wells received medium with no added insulin). Media were removed after 15 min, followed by cell lysis using RIPA buffer with protease and phosphatase inhibitors (Cell Signaling Technology, Danvers, MA). Protein concentrations in cell lysates were determined with a BCA assay kit (Thermo Fisher Scientific) for use in immunoblotting. Cells cultured for q-rtPCR assays (see below) were, after 24 h of serum starvation, treated with either a protein-free medium or medium supplemented with an insulin analog medium for 8 h.
Quantitative real-time PCR transcriptional assay
Mitogenic gene transcription was measured in triplicate by q-rtPCR with samples prepared according to the one-step q-rtPCR kit (Bio-Rad). The following sets of primers (5′–3′) were used for MCF-7 cell-based assays and the following primers (5′–3′) were used: cyclin D1, AATGACCCCGCACGATTTC and TCAGGTTCAGGCCTTGCAC; cyclin G2, ATCGTTTCAAGGCGCACAG and CAACCCCCCTCAGGTATCG; GAPDH, AGCCGAGCCACATCGCT and TGGCAACAATATCCACTTTACCAGAGT; and TFIID, GCACAGGAGCCAAGAGTGAA and TCACAGCTCCCCACCATGTT.
Insulin-stimulated protein phosphorylation
Cell lysates were dissolved in Laemmli buffer (Bio-Rad) with 10% β-mercaptoethanol, heated at 100 °C for 8 min (for MCF-7 p-IR/IR), and then centrifuged at 9300 × g for 1 min (Biofuge A; Baxter Scientific, Soddy Daisy, TN). Samples were loaded into 10% MiniPROTEAN TGX gels (Bio-Rad). Proteins were transferred to polyvinylidene difluoride membrane and then blocked in 5% BSA for 1 h. All antibodies were purchased from Cell Signaling Technology unless otherwise stated. Membranes were incubated overnight at 4 °C with insulin receptor β (4B8) rabbit monoclonal antibody (mAb) or an equal mixture of phospho-insulin receptor β (Tyr1150/1151) rabbit mAb; phospho-insulin receptor (Tyr1158) polyclonal antibody (Thermo Fisher Scientific); phospho-insulin receptor (Tyr1334) polyclonal antibody (Thermo Fisher Scientific); phospho-insulin receptor β (Tyr1345) rabbit mAb; and anti-insulin receptor (phospho-Tyr972) antibody (Abcam, Cambridge, UK). Dilutions for these antibodies were 1:5000 in 5% BSA. After the primary antibody incubation, membranes were washed with 1×TBS with 0.1% Tween 20 and incubated in goat anti-rabbit HRP-conjugated secondary antibody diluted 1:10,000 in 5% BSA for 1–2 h at room temperature. All membranes were washed three times with 1×TBS, 0.1% Tween 20 and incubated in HRP substrate (Merck Millipore, Billerica, MA) for 45 s and developed.
Assessment of fibril formation
Physical stability was assessed by propensity to form fibrils. Insulin or analogs were made 60 μm in phosphate-buffered saline (PBS; pH 7.4) with 0.02% sodium azide and 16 μm ThT. Samples were plated in a Costar® plate (250 μl/well) and incubated at 37 °C with continuous shaking at 1096 cycles/min in a Biotek (Winooski, VT) Synergy H1® plate reader. ThT fluorescence at 480 nm was assessed (following excitation at 450 nm) at 15-min intervals. The time of initial THT fluorescence defined the lag time.
Fibrillation lag times of active [GlyB24]insulin analogs containing Cha or Leu at the B25 position were assessed in relation to control analogs at room temperature in a milder assay designed to simulate a patient-carried insulin pump. Analogs were made 60 μm in PBS containing 0.01% sodium azide as an antimicrobial agent. The insulin solutions were gently rocked at 25 °C in glass vials containing a liquid–air interface. Aliquots, taken at regular intervals, were frozen to enable later analysis of ThT fluorescence (
). WT insulin or analogs were made 0.6 mm in a buffer containing 50 mm Tris-HCl (pH 7.4), 50 mm phenol, 0.2 mm CoCl2, and 1 mm NaSCN. Samples were incubated overnight at room temperature. Spectra (450–700 nm) were obtained to monitor tetrahedral Co2+ coordination with its signature peak absorption band at 574 nm (
). To determine the rate of Co2+ release from the hexamers, metal-ion sequestration was initiated at 25 °C by addition of an aliquot of EDTA (stored at 50 mm at pH 7.4) to a final concentration of 2 mm. Attenuation of the 574-nm absorption band was monitored on a time scale of seconds to hours. Kinetic data were consistent with monoexponential decay and were fit using Kaleidagraph® software allowing for calculation of kinetic half-life (t½).
Assessment of insulin self-assembly
Oligomeric states of the insulin analogs were monitored by HPLC SEC (
). Analogs were made 0.6 mm in PBS. Samples (10 μl) were applied through a Waters 717 autosampler onto a Zenix-C SEC-150 column (Sepax Technologies) with a nominal fractionation range of 0.5–150 kDa. Proteins were fractionated at a flow rate of 1 ml/min using a Waters Binary HPLC system. Elution was monitored at 215 and 280 nm using a dual-λ Waters 2487 absorbance detector. The mobile phase consisted of 10 mm Tris-HCl (pH 7.4) and 140 mm NaCl (
). Data acquisition and processing utilized Waters HPLC Empower software. The column was calibrated for apparent molecular-mass determination by fractionating standard proteins individually on the column (see Fig. S9 legend).
Cell-based proinsulin folding and secretion
HEK293T cells were plated into 6- or 12-well plates 1 day before transfection. A total of 1–2 μg of plasmid DNA was transfected using Lipofectamine (Invitrogen®). At 48 h post-transfection, cells were pulse-labeled with [35S]Cys/Met and chased for 120 min. A proteinase inhibitor mixture was added to cell lysates and chase media. Samples were pre-cleared with Zysorbin and immunoprecipitated with anti-insulin antibodies (
) and protein-A–agarose overnight at 4 °C. Anti-insulin immunoprecipitates were boiled for 5 min in gel sample buffer (1% SDS, 12% glycerol, and 0.0025% Serva Blue in 50 mm Tris (pH 6.8)) and analyzed using Tris–Tricine–urea–SDS-PAGE under nonreducing conditions (
), CMV–Renilla–luciferase plasmid (Promega), and human WT or mutant proinsulin at a DNA ratio of 1:2:5, respectively. At 48 h post-transfection, cell extracts were prepared for the dual-luciferase reporter assay (Promega) with BiP-luciferase normalized to Renilla luciferase activity.
Confocal microscopy of transiently transfected β-cells
INS1 cells were co-transfected with plasmids encoding (a) GFP-tagged proinsulin–WT or mutants and (b) RFP fused with KEDL sequence. At 48 h post-transfection, the cells were fixed with 3.7% formalin in PBS for 20 min, mounted with Prolong Gold with DAPI (Invitrogen), and imaged by epifluorescence in an Olympus FV500 confocal microscope. For immunofluorescence, transfected cells were fixed with 3.7% formalin in PBS for 20 min, permeabilized with TBS containing 0.4% Triton X-100, blocked with TBS containing 3% bovine serum albumin (BSA) and 0.2% Triton X-100, and then stained with primary rabbit anti-calnexin at 4 °C overnight. Thereafter, sections were rinsed and incubated with secondary antibodies conjugated to Alexa Fluor 488 or 568 (Invitrogen). Slides were mounted with Prolong Gold with DAPI (Invitrogen) and imaged by epifluorescence in an Olympus FV500 confocal microscope (
). Both the monomeric NMR structure of insulin (PDB code 2KJJ) and the crystal structure of insulin in complex with the micro-receptor and the IR-A isoform αCT (PDB code 4OGA) were used as templates, modeling only the L1 and αCT IR domains in complex with [GlyB24]insulin. Models were generated both with the alignment against the templates leading to a void generated in the B24 pocket (GlyB24 aligned at the B24 position) and with the B chain register-shift leading to PheB25 occupying the B24 pocket. Glycosylation is accounted for via the use of a single N-linked N-acetyl-d-glucosamine carbohydrate at each of the IR asparagine residues 16, 25, and 111. From the 50 models created, the model with the lowest MODELLER objective function was used in subsequent MD simulations.
MD simulations were performed, as described previously (
) force field. The simulation consisted of an initial steepest descent minimization, a short 50-ps positionally restrained MD holding the protein fixed, and finally unrestrained MD for 250 ns. Each complex was placed within a cubic box extending 10 Å beyond all atoms, with the remaining volume solvated using the TIP3P water molecule with sodium and chloride ions to an ionic strength of 0.1 m. The temperature and pressure of the systems were maintained using the velocity rescaling (
) to account for long-range electrostatics applying a grid width of 1.2 Å and a sixth-order spline interpolation. All bond lengths were constrained with the P-LINCS algorithm, which allowed a time step of 2 fs.
) webservers were used to estimate in silico the effect of the GlyB24 mutation on the interaction between insulin and the IR primary binding site. Cavity analysis was subsequently performed using the trj_cavity tool (
) across all trajectories, defining cavities with a minimum volume of 50 Å3, and then were completely buried (i.e. in all six dimensions surrounding each voxel).
N. K. R., M. L., N. F. P., B. J. S., P. A., and M. A. W. conceptualization; N. K. R. and L. H. data curation; N. K. R., M. L., and L. H. validation; N. K. R., M. L., B. D., Y.-S. C., N. A. S., L. R., J. S., H. G., Y. Y., N. F. P., B. J. S., and F. I.-B. investigation; N. K. R. and M. A. W. writing-original draft; M. L., N. F. P., P. A., F. I.-B., and M. A. W. formal analysis; M. L., N. F. P., J. W., P. A., and F. I.-B. methodology; M. L., Y.-S. C., L. H., N. F. P., B. J. S., P. A., F. I.-B., and M. A. W. writing-review and editing; N. F. P., P. A., F. I.-B., and M. A. W. supervision; N. F. P., P. A., F. I.-B., and M. A. W. funding acquisition.
We thank M. C. Lawrence, J. G. Menting, and V. Pandyarajan for helpful discussion; K. Carr, R. Grabowski, P. Macklis, and M. Swain for assistance with rat studies; L. Whittaker for advice regarding IR-binding assays; and P. DeMeyts (Novo Nordisk) for the gift of 125I-radiolabeled insulin.