Differential 14-3-3 Affinity Capture Reveals New Downstream Targets of Phosphatidylinositol 3-Kinase Signaling

We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d₀/d₄) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d₀/d₄ values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d₀/d₄ scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser¹⁹ of ZNRF2 (RTRAYpS¹⁹GS), phospho-Ser⁹⁰ of SASH1 (RKRRVpS⁹⁰QD), and phospho- Ser⁴⁹³ of lipolysis-stimulated lipoprotein receptor (RPRARpS⁴⁹³LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways.Activated tyrosine kinase receptors generally drive cells to assimilate nutrients; regulate partitioning of the assimilate to make storage polymers and biosynthetic precursors and for energy production; and promote cellular survival, growth, division, movement, and differentiation. From this spectrum, each cell displays a specific subset of responses depending on the hormone, specific receptors, cross-talk from other signaling pathways, metabolic conditions, and cellular complement of effector proteins. For example, insulin stimulates glucose uptake and glycogen synthesis in skeletal muscle, whereas IGF1¹ promotes survival, growth, and proliferation of many cell types (1, 2).Many of these cellular responses are mediated via PI 3-kinase, which generates phosphatidylinositol 3,4,5-trisphosphate, promoting the activation of AGC protein kinases such as PKB/Akt and other signaling components (1, 3). PI 3-kinase is activated by binding to tyrosine-phosphorylated receptors such as the platelet-derived growth factor receptor or via adaptor molecules such as insulin receptor substrates, which are phosphorylated by the activated insulin receptor. Deregulated PI 3-kinase and downstream signaling has been linked to problems with wound healing, immune responses, neurodegeneration, and cardiovascular disease; decreased PI 3-kinase signaling may underlie insulin resistance and type II diabetes; and this pathway is often activated in human tumors (4, 5). To help pinpoint drug targets for these diseases we must define the mechanisms linking PI 3-kinase and other signaling pathways to downstream effectors and understand specificity with respect to different hormone/cell type combinations.Many missing substrates of PI 3-kinase/AGC kinases must be found to explain all the cellular responses to insulin and growth factors (3). Several targets of PI 3-kinase/PKB signaling, including TSC2 (6), PRAS40 (7), AS160 (8), and FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase (9) were identified using the anti-PAS antibody, which loosely recognizes the minimal phosphorylated consensus for PKB, which is RXRXX(pS/pT) where pS is phosphoserine and pT is phosphothreonine. Another helpful feature for identifying new downstream targets is that phosphorylation by PKB sometimes creates binding sites for 14-3-3s, which are dimeric proteins that bind to specific phosphorylated sites on target proteins. Thus PKB promotes the binding of 14-3-3s to proteins including PFKFB2 cardiac PFK-2 (10, 11), BimEL (12), β-catenin (13), p27(Kip1) (14), PRAS40 (7), FOXO1 (15), Miz1 (16), TBC1D4 (AS160 (17, 18), and TBC1D1 (19). Functionally 14-3-3s can trigger changes in the conformations of their targets and alter how targets interact with other proteins. Consistent with 14-3-3/target interactions being important in cellular responses to growth factors and insulin, reagents that compete with targets for binding to 14-3-3s inhibit the IGF1-stimulated increase in the glycolytic stimulator fructose-2,6-bisphosphate (10) and PKB-dependent cell survival (20).Some 14-3-3-binding sites on the above named proteins can also be phosphorylated by other basophilic protein kinases (21). For example, AS160 and TBC1D1 are two related RabGAPs (GTPase-activating protein for Rabs) regulated by multisite phosphorylation that regulate trafficking of GluT4 transporter to the plasma membrane for uptake of glucose. The two 14-3-3-binding sites on AS160 can be phosphorylated by PKB, p90RSK, serum- and glucocorticoid-inducible kinase, and other kinases, whereas one of the 14-3-3-binding sites on TBC1D1 is also a substrate of the energy-sensing kinase AMP-activated protein kinase (17–19). Thus, the relative sensitivity of glucose trafficking to insulin and AMP-activated protein kinase activators in different tissues may depend in part on the distribution of AS160 and TBC1D1. Other insulin-regulated 14-3-3 targets, such as myosin 1C (22), are also convergence points for phosphorylation by more than one AGC and/or Ca²⁺/calmodulin-dependent protein kinase.Here many more proteins than those already identified were found to display 14-3-3 and/or PAS binding signals when the PI 3-kinase pathway was activated in cells against a “background” of other proteins whose 14-3-3 and PAS binding status was unaffected by PI 3-kinase signaling. We aimed to pick out the PI 3-kinase-regulated proteins, which was challenging given the hundreds of 14-3-3 binding partners in mammalian cells (10, 23–27). We used 14-3-3 affinity capture and release, identified phosphopeptides, and devised a quantitative proteomics approach in which 14-3-3-binding proteins from insulin-stimulated versus unstimulated cells were labeled with formaldehyde containing light or heavy isotopes, respectively. Biochemical checking of candidates from these screens, which included proteins with links to diabetes, cancers, and neurodegenerative disorders, confirmed the identification of novel downstream targets of PI 3-kinase, some of which are also convergence points for regulation by MAPK/p90RSK signaling.     

Differential Regulation of Elastic Fiber Formation by Fibulin-4 and -5

Fibulin-4 and -5 are extracellular glycoproteins with essential non-compensatory roles in elastic fiber assembly. We have determined how they interact with tropoelastin, lysyl oxidase, and fibrillin-1, thereby revealing how they differentially regulate assembly. Strong binding between fibulin-4 and lysyl oxidase enhanced the interaction of fibulin-4 with tropoelastin, forming ternary complexes that may direct elastin cross-linking. In contrast, fibulin-5 did not bind lysyl oxidase strongly but bound tropoelastin in terminal and central regions and could concurrently bind fibulin-4. Both fibulins differentially bound N-terminal fibrillin-1, which strongly inhibited their binding to lysyl oxidase and tropoelastin. Knockdown experiments revealed that fibulin-5 controlled elastin deposition on microfibrils, although fibulin-4 can also bind fibrillin-1. These experiments provide a molecular account of the distinct roles of fibulin-4 and -5 in elastic fiber assembly and how they act in concert to chaperone cross-linked elastin onto microfibrils.Fibulins are a family of extracellular glycoproteins containing contiguous calcium-binding epidermal growth factor-like domains (cbEGFs)³ and a characteristic C-terminal fibulin (FC) domain (1–3). Recent studies have revealed that fibulin-4 and -5 are both essential for elastic fiber formation (4–7). Fibulin-4 is widely expressed from early embryogenesis and is necessary for normal vascular, lung, and skin development, since mice that lack fibulin-4 do not form elastic fibers and die perinatally (5). Furthermore, mice with reduced fibulin-4 expression develop aneurysms (8). Fibulin-5 is abundant in the aorta and large arteries during embryogenesis and following vascular injury (9, 10). Lack of fibulin-5 causes a less severe phenotype, with viable homozygous mice, but the elastic fibers in skin, lungs, and aorta are irregular and fragmented (6, 7), and there is altered vascular remodeling (11). These mice models also highlight that fibulin-4 and -5 have non-compensatory roles in elastic fiber formation. Mutations in both molecules can cause cutis laxa, a heritable disorder associated with elastic fiber degeneration leading to sagging skin, vascular tortuosity, and emphysematous lungs (12–15). A third isoform, fibulin-3, may play a minor role in elastic fiber formation, since its deficiency disrupts elastic fibers in Bruch''s membrane of the eye (16) and vaginal tissues (17).Elastic fiber formation is a complex multistep process (18–20). Initial pericellular microassembly of tropoelastin, which may involve the 67-kDa elastin-binding protein receptor, generates elastin globules that are stabilized by desmosine cross-links catalyzed mainly by lysyl oxidase (LOX) but also by LOXL1 (LOX-like 1). These globules are deposited on a fibrillin microfibril template, where they coalesce and undergo further cross-linking to form the elastin core of mature fibers. The ability of fibulin-4 and -5 to bind tropoelastin and fibrillin-1, the major structural component of microfibrils, supports a model in which these fibulins direct elastin deposition on microfibrils (4–7, 21–25). This model does not delineate the unique molecular contributions of fibulin-4 and -5 to elastic fiber formation, but some molecular differences have emerged. Tropoelastin was bound more strongly by fibulin-5 than by fibulin-4, whereas fibulin-5 was at the microfibril-elastin interface, but perichondrial fibulin-4 localized mainly to microfibrils (4).Fibulin-4 null mice offer tantalizing clues to how fibulin-4 contributes to elastic fiber formation (5). They had dramatically reduced (94%) desmosine cross-links despite no change in elastin or LOX expression levels, and electron-dense rodlike structures were prominent within elastin aggregates. Morphologically similar structures seen after chemically inhibiting LOX were previously identified as glycosaminoglycans, which can bind charged free ϵ-amino groups on lysines in tropoelastin (26). However, fibulin-4^+/− mice showed ∼20% increase in desmosine (5). LOX-null mice have phenotypic features similar to those of fibulin-4 null mice, dying perinatally with 60% reduced desmosine cross-links and major abnormalities in vascular and other elastic tissues (27, 28). In contrast, LOXL1-null mice are viable but have reduced desmosine (29), whereas fibulin-5 null mice have a 16% reduction in desmosine cross-links and survive well into adulthood (7). Detection of the LOXL1 pro-domain in fibulin-5 null mice skin but not wild-type skin implicates fibulin-5 in activation of LOXL1 (30).We and others have shown that fibrillin-1 and the microfibrillar protein MAGP-1 can both directly bind tropoelastin (31–34). However, the fibulin-null mice show that the fibrillin-1 interaction with tropoelastin is insufficient to support elastic fiber formation in vivo. Fibulin-5 has been reported to facilitate tropoelastin binding to the N-terminal half of fibrillin-1 (21). A study of elastin polypeptide self-assembly through coacervation and maturation phases showed that, although the N-terminal half of fibrillin-1 increased maturation velocity and droplet clustering, fibulin-4 and -5 both slowed maturation and limited globule growth (35). These studies imply that fibulins and fibrillin-1 act together to regulate elastin accretion on microfibrils.To gain further insights into the contributions of fibulin-4 and -5 to elastic fiber formation, we have delineated how they interact with tropoelastin, LOX, and fibrillin-1. Novel findings are that fibulin-4 directly binds LOX, and this interaction enhances fibulin-4 binding to tropoelastin, thus forming a ternary complex that may be critical for elastin cross-linking. Fibulin-5 can concurrently bind fibulin-4 and tropoelastin, but the interaction of both fibulins with fibrillin-1 strongly inhibits their binding to tropoelastin. These interactions indicate the molecular basis of how fibulins act as chaperones for deposition of elastin onto microfibrils. Our study thus provides a molecular account of the differential roles of fibulins-4 and -5 in elastic fiber formation.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Proteome-wide Substrate Analysis Indicates Substrate Exclusion as a Mechanism to Generate Caspase-7 Versus Caspase-3 Specificity

Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6–P5′ undecapeptide retained complete specificity for caspase-7. The corresponding P6–P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P′ residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4–P1 residues constitute the core cleavage site but that P6, P5, P2′, and P3′ residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7.Caspases, a family of evolutionarily conserved proteases, mediate apoptosis, inflammation, proliferation, and differentiation by cleaving many cellular substrates (1–3). The apoptotic initiator caspases (caspase-8, -9, and -10) are activated in large signaling platforms and propagate the death signal by cleavage-induced activation of executioner caspase-3 and -7 (4, 5). Most of the cleavage events occurring during apoptosis have been attributed to the proteolytic activity of these two executioner caspases, which can act on several hundreds of proteins (2, 3, 6, 7). The substrate degradomes of the two main executioner caspases have not been determined but their identification is important to gaining greater insight in their cleavage specificity and biological functions.The specificity of caspases was rigorously profiled by using combinatorial tetrapeptide libraries (8), proteome-derived peptide libraries (9), and sets of individual peptide substrates (10, 11). The results of these studies indicate that specificity motifs for caspase-3 and -7 are nearly indistinguishable with the canonical peptide substrate, DEVD, used to monitor the enzymatic activity of both caspase-3 and -7 in biological samples. This overlap in cleavage specificity is manifested in their generation of similar cleavage fragments from a variety of apoptosis-related substrates such as inhibitor of caspase-activated DNase, keratin 18, PARP,1 protein-disulfide isomerase, and Rho kinase I (for reviews, see Refs. 2, 3, and 7). This propagated the view that these two caspases have completely redundant functions during apoptosis. Surprisingly, mice deficient in one of these caspases (as well as mice deficient in both) have distinct phenotypes. Depending on the genetic background of the mice, caspase-3-deficient mice either die before birth (129/SvJ) or develop almost normally (C57BL/6J) (12–14). This suggests that dynamics in the genetic background, such as increased caspase-7 expression, compensate for the functional loss of caspase-3 (15). In the C57BL/6J background, caspase-7 single deficient mice are also viable, whereas caspase-3 and -7 double deficient mice die as embryos, further suggesting redundancy (12–14). However, because caspase-3 and -7 probably arose from gene duplication between the Cephalochordata-Vertebrata diversion (16), they might have acquired different substrate specificities during evolution. Caspase-3 and -7 do exhibit different activities on a few arbitrarily identified natural substrates, including BID, X-linked inhibitor of apoptosis protein, gelsolin, caspase-6, ataxin-7, and co-chaperone p23 (17–20). In addition, caspase-3 generally cleaves more substrates during apoptosis than caspase-7 and therefore appears to be the major executioner caspase. Moreover, a recent report describing caspase-1-dependent activation of caspase-7, but not of caspase-3, in macrophages in response to microbial stimuli supports the idea of a non-redundant function for caspase-7 downstream of caspase-1 (21).Commercially available “caspase-specific” tetrapeptide substrates are widely used for specific caspase detection, but they display substantial promiscuity and cannot be used to monitor individual caspases in cells (22, 23). Detecting proteolysis by measuring the release of C-terminal fluorophores, such as 7-amino-4-methylcoumarin (amc), restricts the specificity of these peptide substrates to non-prime cleavage site residues, which may have hampered the identification of specific cleavage events. To address this limitation, a recently developed proteomics technique, called proteomic identification of protease cleavage sites, was used to map both non-prime and prime preferences for caspase-3 and -7 on a tryptic peptide library (9). However, no clear distinction in peptide recognition motifs between caspase-3 and -7 could be observed (9). Because not all classical caspase cleavage sites are processed (7), structural or post-translational higher order constraints are likely involved in steering the cleavage site selectivity. Peptide-based approaches generally overlook such aspects.We made use of the COFRADIC N-terminal peptide sorting methodology (24–26) to profile proteolytic events of caspase-3 and -7 in a macrophage proteome labeled by triple stable isotope labeling by amino acids in cell culture (SILAC), which allowed direct comparison of peak intensities in peptide MS spectra and consequent quantification of N termini that are equally, preferably, or exclusively generated by the action of caspase-3 or -7 (26, 27). We identified 55 cleavage sites in 48 protein substrates, encompassing mutual, preferred, and unique caspase-3 and -7 cleavage sites.     

Evidence of a Role for Phosphatidylinositol 3-Kinase Activation in the Blocking of Apoptosis by Polyomavirus Middle T Antigen

A polyomavirus mutant (315YF) blocked in binding phosphatidylinositol 3-kinase (PI 3-kinase) has previously been shown to be partially deficient in transformation and to induce fewer tumors and with a significant delay compared to wild-type virus. The role of polyomavirus middle T antigen-activated PI 3-kinase in apoptosis was investigated as a possible cause of this behavior. When grown in medium containing 1d-3-deoxy-3-fluoro-myo-inositol to block formation of 3′-phosphorylated phosphatidylinositols, F111 rat fibroblasts transformed by wild-type polyomavirus (PyF), but not normal F111 cells, showed a marked loss of viability with evidence of apoptosis. Similarly, treatment with wortmannin, an inhibitor of PI 3-kinase, stimulated apoptosis in PyF cells but not in normal cells. Activation of Akt, a serine/threonine kinase whose activity has been correlated with regulation of apoptosis, was roughly twofold higher in F111 cells transformed by either wild-type virus or mutant 250YS blocked in binding Shc compared to cells transformed by mutant 315YF. In the same cells, levels of apoptosis were inversely correlated with Akt activity. Apoptosis induced by serum withdrawal in Rat-1 cells expressing a temperature-sensitive p53 was shown to be at least partially p53 independent. Expression of either wild-type or 250YS middle T antigen inhibited apoptosis in serum-starved Rat-1 cells at both permissive and restrictive temperatures for p53. Mutant 315YF middle T antigen was partially defective for inhibition of apoptosis in these cells. The results indicate that unlike other DNA tumor viruses which block apoptosis by inactivation of p53, polyomavirus achieves protection from apoptotic death through a middle T antigen–PI 3-kinase–Akt pathway that is at least partially p53 independent.Programmed cell death occurs during normal development and under certain pathological conditions. In mammalian cells, apoptosis can be induced by a variety of stimuli, including DNA damage (45), virus infection (54, 57), oncogene activation (25), and serum withdrawal (34, 37). Apoptosis can also be blocked by a number of factors, including adenovirus E1B 55- or 19-kDa proteins (9, 16), baculovirus p35 and iap genes (10), Bcl-2 (36, 61), and survival factors (12, 21). DNA tumor viruses have evolved mechanisms that both trigger and inhibit apoptosis. These frequently involve binding and inactivation of tumor suppressor proteins. E7 in some papillomaviruses (22), E1A in adenovirus (31, 43, 64), and large T antigen in simian virus 40 (SV40) (17) bind Rb and/or p300 and lead to upregulation of p53, which is thought to trigger apoptosis in virus-infected cells. The same viruses also inhibit apoptosis by inactivating p53 by various mechanisms (44, 63, 67). In contrast, the mechanism by which polyomavirus interacts with apoptotic pathways in the cell is not known; no direct interaction with p53 by any of the proteins encoded by this virus has been demonstrated (19, 62).The principal oncoprotein of polyomavirus is the middle T antigen. Neoplastic transformation by polyomavirus middle T antigen has as a central feature its association with and activation of members of the Src family of tyrosine kinases p60^c-src (13) and p62^c-yes (42). The major known consequence of these interactions is phosphorylation of middle T antigen on specific tyrosine residues creating binding sites for other signaling proteins. Phosphorylation at tyrosines 250, 315, and 322 promotes binding to Shc (18), the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (59), and phospholipase Cγ-1 (58), respectively. Recognition of multiple signaling pathways emanating from middle T antigen has led to a keen interest in identifying their downstream biochemical effects, which collectively lead to the emergence of neoplastic transformation and presumably underlie the dramatic ability of the virus to induce many kinds of tumors in the mouse.Previous work has shown that the binding of PI 3-kinase to middle T antigen is essential for full transformation of rat fibroblasts in culture (8) and for rapid development of a broad spectrum of tumors in mice (30), for translocation of the GLUT1 transporter (68), and activation of p70 S6 kinase (14). While the mutant 315YF (blocked in PI 3-kinase activation) was able to induce some tumors, it did so at reduced frequencies and with an average latency three times longer than that of either the wild-type virus or a mutant, 250YS, blocked in binding Shc (4, 30). Recent studies have indicated a role of PI 3-kinase in blocking apoptosis in nonviral systems. Growth factor receptors acting through protein tyrosine kinases may prevent apoptosis by activating PI 3-kinase in PC12 cells, T lymphocytes, hematopoietic progenitors, and rat fibroblasts (7, 48, 56, 65, 66). The failure of mutant 315YF to induce full transformation of cells in culture and to induce the rapid development of tumors in mice could therefore be related, at least in part, to a failure to block apoptosis. In this study, we focus on the question of whether middle T antigen–PI 3-kinase interaction is involved in blocking apoptosis in cells transformed by polyomavirus.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.