Biology subjects

Dhayalan, B.

Publications and source records attributed to Dhayalan, B..

6 recordsLinked to original sources

bioRxiv2023

Se-Glargine I. Chemical Synthesis of a Basal Insulin Analog Stabilized by an Internal Diselenide Bridge

Insulin, a small globular protein, has long provided a model for studies of biophysical principles with therapeutic application. The safety and efficacy of insulin replacement therapy for the treatment for diabetes mellitus have been enhanced by protein engineering. Here, we describe the chemical synthesis of a basal insulin analog stabilized by the substitution of an internal cystine (A6-A11) by a diselenide bridge. The studies focused on insulin glargine, the active component of clinical products Lantus(R) and Toujeo(R) (Sanofi). Formulated in solution at pH 4 in the presence of zinc ions, insulin glargine exhibits a shifted isoelectric point (from pH 4.5 to neutral pH) due to a basic extension of the B chain (ArgB31-ArgB32). Subcutaneous injection of such an acidic formulation leads to pH-dependent precipitation of protein-zinc complexes to form a long-lived depot. Pairwise substitution of CysA6 and CysA11 by selenocysteine (Sec; the 21st encoded amino acid) was effected by solid-phase peptide synthesis. The modified A chain also contained substitution of AsnA21 by Gly, introduced in glargine to avoid acid-catalyzed deamidation of the A21 carboxamide group in the formulation. Although classical chain combination of the di-Arg-extended B chain and modified A chain exhibited lower yield than does wild-type chain combination, substantial product was obtained through repeated reactions and successive purification. This strategy exemplifies the rational optimization of protein stability and may be generalizable to diverse disulfide-stabilized proteins of therapeutic interest.

biochemistry↗

bioRxiv2023

Se-Glargine II. Native Function of a Basal Insulin Analog Stabilized by an Internal Diselenide Bridge

Insulin glargine, the active component of basal clinical pharmaceutical formulations Lantus(R) and Toujeo(R) (Sanofi), provides a model for principles of therapeutic protein design. Formulated in solution at pH 4, insulin glargine exhibits a shifted isoelectric point (from pH 5.3 to neutral pH) due to a basic dipeptide B-chain extension (ArgB31-ArgB32). In the first article in this series, we described pairwise substitution of CysA6 and CysA11 by seleno-cysteine (Sec; the 21st encoded amino acid) by solid-phase peptide synthesis. 1H-2H amide proton exchange, as monitored by 1H-NMR spectroscopy, provides evidence that substitution of internal cystine A6-A11 by a diselenide bridge stabilizes the protein and damps segmental conformational fluctuations. Further, this analog and its major metabolites M1 and M2 (respectively denoting proteolytic derivatives lacking ArgB31-ArgB32 or ThrB30-ArgB31-ArgB32) exhibit native hormonal activity in mammalian cell-based assays measuring dose-dependent autophosphorylation of the insulin receptor (pIR/IR ratio) and metabolic gene regulation in human liver-derived HepG2 cells. The internal diselenide bridge also did not alter respective baseline mitogenicities of insulin glargine or its proteolytic products as evaluated by a qPCR-based assay of the balance between proliferative and antiproliferative cyclin gene expression; the assays employed L6 myoblasts over-expressing mitogenic IR isoform A. Given such native function, shelf life--and hence global access to insulin in the developing world--may be enhanced by stabilizing diselenide chemistry.

biochemistry↗

bioRxiv2022

Stabilization of a Protein by a Single Halogen-Based Aromatic Amplifier

The utility of halogenation in protein design is investigated by a combination of quantitative atomistic simulations and experiment. The approach is applied to insulin, a small, therapeutically relevant domain amenable to simulation and semi-synthesis. In a singly halogenated aromatic ring, the simulations predicted regiospecific inductive effects to modulate multiple surrounding electrostatic (weakly polar) interactions, thereby amplifying changes in thermodynamic stability. In accordance with the simulations, stabilization of insulin is demonstrated by single halogen atoms at the ortho position of an invariant phenylalanine (2-F-PheB24, 2-Cl-PheB24 and 2-Br-PheB24; {Delta}{Delta}Gu = -0.5 to -1.0 kcal/mol) located at the edge of a protein crevice. Corresponding meta and para substitutions have negligible effects. The ortho-modified insulin analogs exhibit enhanced resistance to fibrillation above room temperature and retain biological activity in mammalian cells and in a rat model of diabetes mellitus. Consequently, halogen-based stabilization of insulin and other therapeutic proteins may provide a biophysical strategy to circumvent the requirement for a distribution "cold chain" in the developing world and enhance the shelf life of pharmaceutical formulations.

biochemistry↗

bioRxiv2021

Age of diabetes onset in the mutant proinsulin syndrome correlates with mutational impairment of protein foldability and stability

Diverse heterozygous mutations in the human insulin gene cause a monogenic diabetes mellitus (DM) syndrome due to toxic misfolding of the variant proinsulin. Whereas mutations that add or remove cysteines (thereby leading to an odd number of thiol groups) generally lead to neonatal-onset DM, non-Cys-related mutations can be associated with a broad range of ages of onset. Here, we compare two mutations at a conserved position in the central B-chain -helix: one neonatal in DM onset (ValB18[->]Gly) and the other with onset delayed until adolescence (AlaB18). The substitutions were introduced within a 49-residue single-chain insulin precursor optimized for folding efficiency (Zaykov, A., et al. ACS Chem. Biol. 9, 683-91 (2014)). Although mutations are each unfavorable, GlyB18 (a) more markedly perturbs DesDi folding efficiency in vitro than does AlaB18 and (b) more severely induces endoplasmic reticulum (ER) stress in cell-based studies of the respective proinsulin variants. In corresponding two-chain hormone analogs, GlyB18 more markedly perturbs structure, function and thermodynamic stability than does AlaB18. Indeed, the GlyB18-insulin analog forms a molten globule with attenuated -helix content whereas the AlaA18 analog retains a nativelike cooperative structure with reduced free energy of unfolding ({Delta}{Delta}Gu 1.2({+/-}0.2) kcal/mole relative to ValB18 parent). We propose that mutations at B18 variably impede nascent pairing of CysB19 and CysA20 to an extent correlated with perturbed core packing once native disulfide pairing is achieved. Differences in age of disease onset (neonatal or adolescent) reflect relative biophysical perturbations (severe or mild) of an obligatory on-pathway protein folding intermediate.

biochemistry↗

bioRxiv2021

Distinct states of proinsulin misfolding in MIDY

A 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 {beta}-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.

biochemistry↗

bioRxiv2019

Role of Proinsulin Self-Association in Mutant INS gene-induced Diabetes of Youth

Abnormal interactions between misfolded mutant and wild-type (WT) proinsulin 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, proinsulin-WT dimerizes in the ER. Here, we suggest that the normal proinsulin-proinsulin contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that proinsulin Tyr-B16, which is a key residue in normal proinsulin dimerization, helps confer dominant-negative behavior of MIDY mutant proinsulin-C(A7)Y. Substitutions of Tyr-B16 with ether Ala, Asp, or Pro in proinsulin-C(A7)Y each decrease the abnormal interactions between the MIDY mutant and proinsulin-WT, rescuing proinsulin-WT export, limiting ER stress, and increasing insulin production in {beta}-cells and human islets. This study reveals the first evidence indicating that noncovalent proinsulin-proinsulin contact initiates dominant-negative behavior of misfolded proinsulin, pointing to a novel therapeutic target to enhance bystander proinsulin export for increased insulin production.

cell biology↗