Browsing by Author "Dhayalan, Balamurugan"
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Item Distinct states of proinsulin misfolding in MIDY(Springer, 2021-08) Haataja, Leena; Arunagiri, Anoop; Hassan, Anis; Regan, Kaitlin; Tsai, Billy; Dhayalan, Balamurugan; Weiss, Michael A.; Liu, Ming; Arvan, Peter; Biochemistry and Molecular Biology, School of MedicineA precondition for efficient proinsulin export from the endoplasmic reticulum (ER) is that proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) "interchain" disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a "lose-A6/A11" mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed proinsulin disulfide bond formation. Here, we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep + 2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic β-cells. Three of these mutants, however, must disrupt the Cys(A6)-Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of proinsulin folding and ER export, because when introduced into the proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type proinsulin. A growing molecular understanding of proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.Item Evolution of insulin at the edge of foldability and its medical implications(National Academy of Sciences, 2020-11-24) Rege, Nischay K.; Liu, Ming; Yang, Yanwu; Dhayalan, Balamurugan; Wickramasinghe, Nalinda P.; Chen, Yen-Shan; Rahimi, Leili; Guo, Huan; Haataja, Leena; Sun, Jinhong; Ismail-Beigi, Faramarz; Phillips, Nelson B.; Arvan, Peter; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineProteins 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.Item Insertion of a Synthetic Switch Into Insulin Provides Metabolite-Dependent Regulation of Hormone-Receptor Activation(Endocrine Society, 2021-05-03) Chen, Yen-Shan; Gleaton, Jeremy; Yang, Yanwu; Dhayalan, Balamurugan; Phillips, Nelson B.; Liu, Yule; Broadwater, Laurie; Jarosinski, Mark; Chatterjee, Deepak; Lawrence, Michael C.; Hattier, Thomas; Michael, Dodson M.; Weiss, Michael Aaron; Biochemistry and Molecular Biology, School of MedicineInsulin signaling requires conformational change: whereas the free hormone and its receptor each adopt autoinhibited conformations, their binding leads to large-scale structural reorganization. To test the coupling between insulin’s “opening” and receptor activation, we inserted an artificial ligand-dependent switch into insulin. Ligand binding disrupts an internal tether designed to stabilize the hormone’s native closed and inactive conformation, thereby enabling productive receptor engagement. This scheme exploited a diol sensor (meta-fluoro-phenylboronic acid at GlyA1) and internal diol (3,4-dihydroxybenzoate at LysB28). The sensor recognizes monosaccharides (fructose > glucose). Studies of insulin signaling in human hepatoma-derived cells (HepG2) demonstrated fructose-dependent receptor autophosphorylation leading to appropriate downstream signaling events, including a specific kinase cascade and metabolic gene regulation (gluconeogenesis and lipogenesis). Addition of glucose (an isomeric ligand with negligible sensor affinity) did not activate the receptor. Similarly, metabolite-regulated signaling was not observed in control studies of (i) an unmodified insulin analog or (ii) an analog containing a diol sensor in the absence of internal tethering. Although as expected CD-detected secondary structure was unaffected by ligand binding, heteronuclear NMR studies revealed subtle local and nonlocal monosaccharide-dependent changes in structure. Insertion of a synthetic switch into insulin has thus demonstrated coupling between hinge-opening and holoreceptor signaling. In addition to this basic finding, our results provide proof of principle for a mechanism-based metabolite-responsive insulin. In particular, replacement of the present fructose sensor by an analogous glucose sensor may enable translational development of a “smart” insulin analog designed to mitigate risk of hypoglycemia in the treatment of diabetes mellitus.Item Insertion of a synthetic switch into insulin provides metabolite-dependent regulation of hormone–receptor activation(National Academy of Sciences, 2021) Chen, Yen-Shan; Gleaton, Jeremy; Yang, Yanwu; Dhayalan, Balamurugan; Phillips, Nelson B.; Liu, Yule; Broadwater, Laurie; Jarosinski, Mark A.; Chatterjee, Deepak; Lawrence, Michael C.; Hattier, Thomas; Michael, M. Dodson; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineLigand-dependent conformational switches are ubiquitous in biological macromolecules, from allosteric proteins to RNA riboswitches. Molecular design of artificial switches provides a general strategy to test relationships between macromolecular structure and function. The present study exploited recent structures of complexes between an ancestral signaling protein (insulin) and the ectodomain of its cellular receptor to insert a metabolite-regulated switch into the hormone. Whereas binding of ligands often stabilizes structure, this design envisioned metabolite-dependent “opening” of a closed, inactive insulin conformation. Assessment of hormone-directed receptor autophosphorylation and a downstream signaling cascade in liver-derived cells demonstrated that binding of metabolite (a monosaccharide) enabled hormonal signaling. These results suggest a mechanism-based strategy to design “smart” glucose-responsive analogs to more safely treat insulin-dependent diabetes mellitus.Item New Horizons: Next-Generation Insulin Analogues: Structural Principles and Clinical Goals(The Endocrine Society, 2022) Jarosinski, Mark A.; Chen, Yen-Shan; Varas, Nicolás; Dhayalan, Balamurugan; Chatterjee, Deepak; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineDesign of “first-generation” insulin analogues over the past 3 decades has provided pharmaceutical formulations with tailored pharmacokinetic (PK) and pharmacodynamic (PD) properties. Application of a molecular tool kit—integrating protein sequence, chemical modification, and formulation—has thus led to improved prandial and basal formulations for the treatment of diabetes mellitus. Although PK/PD changes were modest in relation to prior formulations of human and animal insulins, significant clinical advantages in efficacy (mean glycemia) and safety (rates of hypoglycemia) were obtained. Continuing innovation is providing further improvements to achieve ultrarapid and ultrabasal analogue formulations in an effort to reduce glycemic variability and optimize time in range. Beyond such PK/PD metrics, next-generation insulin analogues seek to exploit therapeutic mechanisms: glucose-responsive (“smart”) analogues, pathway-specific (“biased”) analogues, and organ-targeted analogues. Smart insulin analogues and delivery systems promise to mitigate hypoglycemic risk, a critical barrier to glycemic control, whereas biased and organ-targeted insulin analogues may better recapitulate physiologic hormonal regulation. In each therapeutic class considerations of cost and stability will affect use and global distribution. This review highlights structural principles underlying next-generation design efforts, their respective biological rationale, and potential clinical applications.Item Peptide Model of the Mutant Proinsulin Syndrome. I. Design and Clinical Correlation(Frontiers Media, 2022-03-01) Dhayalan, Balamurugan; Glidden, Michael D.; Zaykov, Alexander N.; Chen, Yen-Shan; Yang, Yanwu; Phillips, Nelson B.; Ismail-Beigi, Faramarz; Jarosinski, Mark A.; DiMarchi, Richard D.; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineThe mutant proinsulin syndrome is a monogenic cause of diabetes mellitus due to toxic misfolding of insulin's biosynthetic precursor. Also designated mutant INS-gene induced diabetes of the young (MIDY), this syndrome defines molecular determinants of foldability in the endoplasmic reticulum (ER) of β-cells. Here, we describe a peptide model of a key proinsulin folding intermediate and variants containing representative clinical mutations; the latter perturb invariant core sites in native proinsulin (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser). The studies exploited a 49-residue single-chain synthetic precursor (designated DesDi), previously shown to optimize in vitro efficiency of disulfide pairing. Parent and variant peptides contain a single disulfide bridge (cystine B19-A20) to provide a model of proinsulin's first oxidative folding intermediate. The peptides were characterized by circular dichroism and redox stability in relation to effects of the mutations on (a) in vitro foldability of the corresponding insulin analogs and (b) ER stress induced in cell culture on expression of the corresponding variant proinsulins. Striking correlations were observed between peptide biophysical properties, degree of ER stress and age of diabetes onset (neonatal or adolescent). Our findings suggest that age of onset reflects the extent to which nascent structure is destabilized in proinsulin's putative folding nucleus. We envisage that such peptide models will enable high-resolution structural studies of key folding determinants and in turn permit molecular dissection of phenotype-genotype relationships in this monogenic diabetes syndrome. Our companion study (next article in this issue) employs two-dimensional heteronuclear NMR spectroscopy to define site-specific perturbations in the variant peptides.Item Peptide Model of the Mutant Proinsulin Syndrome. II. Nascent Structure and Biological Implications(Frontiers Media, 2022-03-01) Yang, Yanwu; Glidden, Michael D.; Dhayalan, Balamurugan; Zaykov, Alexander N.; Chen, Yen-Shan; Wickramasinghe, Nalinda P.; DiMarchi, Richard D.; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineToxic misfolding of proinsulin variants in β-cells defines a monogenic diabetes syndrome, designated mutant INS-gene induced diabetes of the young (MIDY). In our first study (previous article in this issue), we described a one-disulfide peptide model of a proinsulin folding intermediate and its use to study such variants. The mutations (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser) probe residues conserved among vertebrate insulins. In this companion study, we describe 1H and 1H-13C NMR studies of the peptides; key NMR resonance assignments were verified by synthetic 13C-labeling. Parent spectra retain nativelike features in the neighborhood of the single disulfide bridge (cystine B19-A20), including secondary NMR chemical shifts and nonlocal nuclear Overhauser effects. This partial fold engages wild-type side chains LeuB15, LeuA16 and PheB24 at the nexus of nativelike α-helices α1 and α3 (as defined in native proinsulin) and flanking β-strand (residues B24-B26). The variant peptides exhibit successive structural perturbations in order: parent (most organized) > SerB24 >> ProA16 > ProB15 (least organized). The same order pertains to (a) overall α-helix content as probed by circular dichroism, (b) synthetic yields of corresponding three-disulfide insulin analogs, and (c) ER stress induced in cell culture by corresponding mutant proinsulins. These findings suggest that this and related peptide models will provide a general platform for classification of MIDY mutations based on molecular mechanisms by which nascent disulfide pairing is impaired. We propose that the syndrome's variable phenotypic spectrum-onsets ranging from the neonatal period to later in childhood or adolescence-reflects structural features of respective folding intermediates.Item Reassessment of an Innovative Insulin Analogue Excludes Protracted Action yet Highlights Distinction between External and Internal Diselenide Bridges(Wiley, 2020-04-09) Dhayalan, Balamurugan; Chen, Yen-Shan; Phillips, Nelson B.; Swain, Mamuni; Rege, Nischay; Mirsalehi, Ali; Jarosinski, Mark; Ismail-Beigi, Faramarz; Metanis, Norman; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineLong-acting insulin analogues represent the most prescribed class of therapeutic proteins. An innovative design strategy was recently proposed: diselenide substitution of an external disulfide bridge. This approach exploited the distinctive physicochemical properties of selenocysteine (U). Relative to wild type (WT), Se-insulin[C7UA , C7UB ] was reported to be protected from proteolysis by insulin-degrading enzyme (IDE), predicting prolonged activity. Because of this strategy's novelty and potential clinical importance, we sought to validate these findings and test their therapeutic utility in an animal model of diabetes mellitus. Surprisingly, the analogue did not exhibit enhanced stability, and its susceptibility to cleavage by either IDE or a canonical serine protease (glutamyl endopeptidase Glu-C) was similar to WT. Moreover, the analogue's pharmacodynamic profile in rats was not prolonged relative to a rapid-acting clinical analogue (insulin lispro). Although [C7UA , C7UB ] does not confer protracted action, nonetheless its comparison to internal diselenide bridges promises to provide broad biophysical insight.Item “Register-shift” insulin analogs uncover constraints of proteotoxicity in protein evolution(Elsevier, 2020-03-06) Rege, Nischay K.; Liu, Ming; Dhayalan, Balamurugan; Chen, Yen-Shan; Smith, Nicholas A.; Rahimi, Leili; Sun, Jinhong; Guo, Huan; Yang, Yanwu; Haataja, Leena; Phillips, Nelson F. B.; Whittaker, Jonathan; Smith, Brian J.; Arvan, Peter; Ismail-Beigi, Faramarz; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineGlobular 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.Item Role of Proinsulin Self-Association in Mutant INS Gene–Induced Diabetes of Youth(American Diabetes Association, 2020-05) Sun, Jinhong; Xiong, Yi; Li, Xin; Haataja, Leena; Chen, Wei; Mir, Saiful A.; Lv, Li; Madley, Rachel; Larkin, Dennis; Anjum, Arfah; Dhayalan, Balamurugan; Rege, Nischay; Wickramasinghe, Nalinda P.; Weiss, Michael A.; Itkin-Ansari, Pamela; Kaufman, Randal J.; Ostrov, David A.; Arvan, Peter; Liu, Ming; Biochemistry and Molecular Biology, School of MedicineAbnormal interactions between misfolded mutant and wild-type (WT) proinsulin (PI) in the endoplasmic reticulum (ER) drive the molecular pathogenesis of mutant INS gene-induced diabetes of youth (MIDY). How these abnormal interactions are initiated remains unknown. Normally, PI-WT dimerizes in the ER. Here, we suggest that the normal PI-PI contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that PI B-chain tyrosine-16 (Tyr-B16), which is a key residue in normal PI dimerization, helps confer dominant-negative behavior of MIDY mutant PI-C(A7)Y. Substitutions of Tyr-B16 with either Ala, Asp, or Pro in PI-C(A7)Y decrease the abnormal interactions between the MIDY mutant and PI-WT, rescuing PI-WT export, limiting ER stress, and increasing insulin production in β-cells and human islets. This study reveals the first evidence indicating that noncovalent PI-PI contact initiates dominant-negative behavior of misfolded PI, pointing to a novel therapeutic target to enhance PI-WT export and increase insulin production.