Reassessment of an Innovative Insulin Analogue Excludes Protracted Action yet Highlights Distinction between External and Internal Diselenide Bridges

Long-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

Utilizing Copper-Mediated Deprotection of Selenazolidine for Cyclic Peptides Synthesis

Selenazoliline (Sez) was originally developed as a masking form of selenocysteine (Sec) for the chemical synthesis of challenging proteins. Here we utilize Sez and our recent reported copper(II)-mediated deprotection for the synthesis of cyclic peptides. This approach allows deprotection, cyclization and deselenization in one-pot, providing several different cyclic peptides in good yields. In addition, the Sec can also be retained, which enhance the oxidative folding of disulfide-rich cyclic proteins, such as the case of Kalata S. </p

Copper-mediated selenazolidine deprotection enables one-pot chemical synthesis of challenging proteins

While chemical protein synthesis (CPS) has granted access to challenging proteins, synthesis of longer proteins is often limited by low abundance or non-strategic placement of cysteine (Cys) residues, essential for native chemical ligations (NCL), as well as multiple purification and isolation steps. Selective deselenization and one-pot CPS serve as key technologies to circumvent these issues. Herein, we describe the one-pot total synthesis of human thiosulfate: glutathione sulfurtransferase (TSTD1), a 115-residue protein with a single Cys residue at its active site, and its seleno-analogue. WT-TSTD1 was synthesized in a C-to-N synthetic approach employing multiple NCL reactions, Cu(II)-mediated deprotection of selenazolidine (Sez), and chemoselective deselenization, all in one-pot. In addition, the protein’s seleno analogue (Se-TSTD1), in which the active site Cys is replaced with selenocysteine, was synthesized with a kinetically controlled ligation in a one-pot, N-to-C synthetic approach. TSTD1’s one-pot synthesis was made possible by the newly reported, rapid, and facile copper-mediated selenazolidine deprotection that can be accomplished in one minute. Finally, catalytic activity of the two proteins indicated that Se-TSTD1 possessed only four-fold lower activity than WT-TSTD1 as a thiosulfate: glutathione sulfurtransferase, suggesting that selenoproteins can have physiologically comparable sulfutransferase activity as their cysteine counterparts. </p

Chemoselective Copper-Mediated Radical Modification of Selenocysteines in Peptides and Proteins

Highly valuable bioconjugated molecules must be synthesized through efficient, chemoselective chemical modifications of peptides and proteins. Herein we report the chemoselective modification of peptides and proteins via a reaction between selenocysteine residues and aryl/alkyl radicals. In situ radical generation from hydrazine substrates and copper ions proceeds rapidly in neat aqueous buffer at near neutral pH (5-8), providing a variety of Se-modified linear and cyclic peptides and proteins conjugated to aryl and alkyl molecules, as well as to affinity label tag (biotin). This chemistry opens a new avenue for chemical protein modifications

Harnessing selenocysteine reactivity for oxidative protein folding

Although oxidative folding of disulfide-rich proteins is often sluggish, this process can be significantly enhanced by targeted replacement of cysteines with selenocysteines. In this study, we examined the effects of a selenosulfide and native versus nonnative diselenides on the folding rates and mechanism of bovine pancreatic trypsin inhibitor. Our results show that such sulfur-to-selenium substitutions alter the distribution of key folding intermediates and enhance their rates of interconversion in a context-dependent manner.ISSN:2041-6520ISSN:2041-653

One-Pot Chemical Protein Synthesis Utilizing Fmoc-Masked Selenazolidine to Address the Redox Functionality of Human Selenoprotein F

Human SELENOF is an endoplasmic reticulum (ER) selenoprotein that contains the redox active motif CXU (C is cysteine and U is selenocysteine), resembling the redox motif of thiol-disulfide oxidoreductases (CXXC). Like other selenoproteins, the challenge in accessing SELENOF has somewhat limited its full biological characterization thus far. Here we present the one-pot chemical synthesis of the thioredoxin-like domain of SELENOF, highlighted by the use of Fmoc-protected selenazolidine, native chemical ligations and deselenization reactions. The redox potential of the CXU motif, together with insulin turbidimetric assay suggested that SELENOF may catalyze the reduction of disulfides in misfolded proteins. Furthermore, we demonstrate that SELENOF is not a protein disulfide isomerase (PDI)-like enzyme, as it did not enhance the folding of the two protein models; bovine pancreatic trypsin inhibitor and hirudin. These studies suggest that SELENOF may be responsible for reducing the non-native disulfide bonds of misfolded glycoproteins as part of the quality control system in the ER

Effect of Tubulin Self-Association on GTP Hydrolysis and Nucleotide Exchange Reactions

Tubulin nucleation, microtubule (MT) assembly, stability, and dynamics depend on GTP hydrolysis and nucleotide exchange reactions. We investigated how the self-association of isolated tubulin dimers affects the rate of GTP hydrolysis and the equilibrium of nucleotide exchange. We used HPLC to determine the concentrations of GDP and GTP and thereby the GTPase activity of SEC-eluted tubulin dimers in assembly buffer solution, free of glycerol and tubulin aggregates. When GTP hydrolysis was negligible, the nucleotide exchange mechanism was studied using HPLC for determining the concentrations of tubulin-free and tubulin-bound GTP and GDP and by SAXS and cryo-TEM. We observed no GTP hydrolysis below the critical conditions for MT assembly, despite the assembly of tubulin 1D curved oligomers and single rings, showing that their assembly did not involve GTP hydrolysis under our conditions. Under conditions enabling spontaneous slow MT assembly, a slow pseudo-first-order GTP hydrolysis kinetics was detected, limited by the rate of MT assembly. Nucleotide exchange depended on the total tubulin concentration and the molar ratio between tubulin-free GDP and GTP. We used a thermodynamic model of isodesmic tubulin self-association, terminated by the formation of tubulin single-rings to calculate, at each tubulin concentration, the distributions of single rings, 1D oligomers, and free dimers, and thereby the molar fractions of dimers with exposed and buried nucleotide exchangeable sites (E-sites). Our analysis shows that the GDP to GTP exchange reaction equilibrium constant was an order-of-magnitude larger for tubulin dimers with exposed E-sites than for assembled dimers with buried E-sites

A targeted approach for the synthesis of multi-phosphorylated peptides: a tool for studying the role of phosphorylation patterns in proteins

ISSN:1477-0520ISSN:1477-053