The role of assembly in insulin's biosynthesis

Dodson, Guy; Steiner, D.

doi:10.1016/s0959-440x(98)80037-7

Cited by 481 publications

(522 citation statements)

References 19 publications

Supporting

Mentioning

511

Contrasting

Unclassified

Order By: Relevance

“…This observation rationalizes (i) the high activity of an Ala B26 analog (36), (ii) the dispensability of Tyr B26 in truncated analogs (37), and (iii) that whereas holoreceptor substitution Arg14Ala (near both Tyr B26 and the B25 main chain) impairs insulin binding >10 3 -fold, Ala substitution of Asp12 (in contact only with Tyr B26 ) impairs hormone binding by only 6-fold (33). We suggest that the conservation of Tyr B26 (2) arises not from its role in receptor binding but instead from its contribution to proinsulin folding (38) and insulin self-assembly (2,39). Indeed, in the free hormone, Tyr B26 inserts within a conserved nonpolar interchain crevice (2); on μIR binding, this crevice is occupied by key αCT side chains His710 and Phe714 (11).…”

Section: Discussionmentioning

confidence: 84%

“…The marked susceptibility of such rodents to β-cell dysfunction when fed a diet high in carbohydrates (especially free sugars, which are almost absent in its natural plant-based diet) may reflect both extracellular-and intracellular proteotoxicity, respectively, due to amyloidogenesis and impaired folding of the divergent proinsulin-the latter leading to ER stress as in human neonatal DM (52). The anomalous molecular features of degu insulin may thus honor in the breach our suggestion that the closed conformation of insulin and its stable self-assembly in secretory vesicles (2,39) evolved to protect the β cell from proteotoxicity. Fig.…”

Section: Discussionmentioning

confidence: 96%

See 1 more Smart Citation

Protective hinge in insulin opens to enable its receptor engagement

Menting

Yang

Chan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

147

257

View full text Add to dashboard Cite

Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal Bchain segment, critical for protective self-assembly in β cells and receptor binding at target tissues. Insight may be obtained from truncated "microreceptors" that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 β-turn, coupling reorientation of Phe B24 to a 60°rotation of the B25-B28 β-strand away from the hormone core to lie antiparallel to the receptor's L1-β 2 sheet. Opening of this hinge enables conserved nonpolar side chains (Ile A2 , Val A3 , Val B12 , Phe B24 , and Phe B25 ) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptorbound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.diabetes mellitus | signal transduction | receptor tyrosine kinase | metabolism | protein structure H ow insulin engages the insulin receptor has inspired speculation ever since the structure of the free hormone was determined by Hodgkin and colleagues in 1969 (1, 2). Over the ensuing decades, anomalies encountered in studies of analogs have suggested that the hormone undergoes a conformational change on receptor binding: in particular, that the C-terminal β-strand of the B chain (residues B24-B30) releases from the helical core to expose otherwise-buried nonpolar surfaces (the detachment model) (3-6). Interest in the B-chain β-strand was further motivated by the discovery of clinical mutations within it associated with diabetes mellitus (DM) (7). Analysis of residuespecific photo-cross-linking provided evidence that both the detached strand and underlying nonpolar surfaces engage the receptor (8).The relevant structural biology is as follows. The insulin receptor is a disulfide-linked (αβ) 2 receptor tyrosine kinase (Fig. 1A), the extracellular α-subunits together binding a single insulin molecule with high affinity (9). Involvement of the two α-subunits is asymmetric: the primary insulin-binding site (site 1*) comprises the central β-sheet (L1-β 2 ) of the first leucine-rich repeat domain (L1) of one α-subunit and the partially helical Cterminal segment (αCT) of the other α-subunit (Fig. 1A) (10). Such binding initiates conformational changes leading to transphosphorylation of the β-subunits' intracellular tyrosine kinase (TK) domains. Structures of wild-type (WT) insulin (or analogs) bound to extracellular receptor fragments were recently...

show abstract

Section: Discussionmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 96%

Protective hinge in insulin opens to enable its receptor engagement

Menting

Yang

Chan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

147

257

View full text Add to dashboard Cite

show abstract

“…OE, insulin aspart precursor AAK ; ؉, insulin aspart precursor Ϫ (minus indicates a direct connection between B29 and A1); f, insulin aspart precursor LWK ; , insulin aspart precursor Ϫ with the additional amino acid substitutions Glu B10 , Asp B28 , His A8 , and Glu A14 ; q, insulin aspart precursor EWK ; and ࡗ, insulin aspart precursor EWK with the additional amino acid substitutions Glu B10 , Asp B28 , His A8 , and Glu A14 . The insulin aspart precursors, except insulin aspart precursor AAK , featured a N-terminal extension with the sequence E(EA) 3 PK that improved Kex2 endoprotease processing during secretion (see "Experimental Procedures" for further details).…”

Section: Figmentioning

confidence: 99%

“…However, many biochemical and structural properties of insulin and proinsulin have evolved in response to differential requirements of biosynthesis, processing, transport, and storage in the ␤-cells of Langerhans islets (2,3). Importantly, insulin and insulin analogues are readily adapted for expression in yeast as single-chain proinsulin-like precursors lacking the connecting peptide in the following configuration: amino acid residues 1-29 of the B-chain connected to the A-chain by a short removable mini-C-peptide and fused to the yeast prepro-␣ factor through a single dibasic cleavage site (secretory expression of insulin in yeast and in vitro enzymatic conversion of the precursor to insulin has recently been reviewed (4,5)).…”

mentioning

confidence: 99%

Engineering-enhanced Protein Secretory Expression in Yeast with Application to Insulin

Kjeldsen

Ludvigsen

Diers

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

Adaptation to efficient heterologous expression is a prerequisite for recombinant proteins to fulfill their clinical and biotechnological potential. We describe a rational strategy to optimize the secretion efficiency in yeast of an insulin precursor by structure-based engineering of the folding stability. The yield of a fast-acting insulin analogue (Asp B28 ) expressed in yeast was enhanced 5-fold by engineering a specific interaction between an aromatic amino acid in the connecting peptide and a phenol binding site in the hydrophobic core of the molecule. This insulin precursor is characterized by significantly enhanced folding stability. The improved folding properties enhanced the secretion efficiency of the insulin precursor from 10 to 50%. The precursor remains fully in vitro convertible to mature fast-acting insulin.Administration of the two-chain 51-amino acid peptide hormone insulin is life sustaining for many of the 150 million diabetic patients in the world. Classical pancreatic extraction results in 10 -15 mg of insulin per pancreas and the number of porcine or bovine pancreases to produce current requirements of insulin (in excess of 7 metric tons) is simply not available. Consequently, a substantial fraction of insulin is today produced by eukaryotic secretory expression in the yeast Saccharomyces cerevisiae. Recently, second generation fast-acting genetically engineered insulin analogues have been engineered for improved diabetes therapy. This is exemplified by the clinically relevant fast-acting insulin, which features an amino acid substitution in position 28 of the B-chain from proline to aspartic acid and is used for treatment of diabetes mellitus (1).However, many biochemical and structural properties of insulin and proinsulin have evolved in response to differential requirements of biosynthesis, processing, transport, and storage in the ␤-cells of Langerhans islets (2, 3). Importantly, insulin and insulin analogues are readily adapted for expression in yeast as single-chain proinsulin-like precursors lacking the connecting peptide in the following configuration: amino acid residues 1-29 of the B-chain connected to the A-chain by a short removable mini-C-peptide and fused to the yeast prepro-␣ factor through a single dibasic cleavage site (secretory expression of insulin in yeast and in vitro enzymatic conversion of the precursor to insulin has recently been reviewed (4, 5)). Removal of the prepro-␣ factor by endoproteolytic cleavage by the Kex2 endoprotease in a late Golgi compartment generates a singlechain insulin precursor with self-association properties and structure essentially similar to that of human two-chain insulin (6, 7).Proteins exit the lumen of the endoplasmic reticulum (ER) 1 after folding, and frequently this is a rate-limiting step in secretion (8 -10). Herein we describe an improvement in eukaryotic secretion efficiency with application to insulin, using a strategy of structure-based rational engineering under the assumption that a more stably folded precursor molecule is ...

show abstract

“…It connects the A and B chains of insulin in the precursor molecule proinsulin, which is stored in secretory granules of the pancreatic b-cell (1-3). C-peptide facilitates the formation of the correct secondary and tertiary structure of the hormone during insulin biosynthesis (3,4). C-peptide and insulin are secreted in equimolar amounts; however, C-peptide does not undergo significant hepatic metabolism.…”

Section: Introductionmentioning

confidence: 99%

Diagnostic performance of the ARCHITECT C-Peptide immunoassay

Schultess¹,

Duren²,

Martens³

et al. 2009

Clinical Chemistry and Laboratory Medicine

View full text Add to dashboard Cite

Background: Measurement of C-peptide under standardized conditions provides a sensitive and wellestablished assessment of b-cell function. We describe the analytical and clinical validation of an automated, microparticle-based chemiluminescent immunoassay method. The assay is designed to measure C-peptide in human serum, plasma and urine. Methods: Assay performance characteristics such as precision and recovery were measured according to protocols established by the Clinical Laboratory Standards Institute (CLSI). A reference range study was conducted. Analytical sensitivity and specificity, interfering substances, recovery, and linearity studies were performed. Method comparison, against the ADVIA Centaur C-Peptide assay (Siemens), was evaluated with clinical specimens from patients with abnormal insulin secretion. Results: The detection limit for this assay was 0.01 ng/mL. Functional sensitivity (inter-assay imprecision F20%) was 0.015 ng/mL at a coefficient of variation (%CV) of 11.2%. Total imprecision was below 6.5% CV. The assay was linear upon dilution. Comparison with the ADVIA Centaur C-Peptide assay yielded a correlation coefficient (r) of 0.99. Conclusions:The ARCHITECT C-Peptide assay measures C-peptide rapidly, accurately, and precisely in human serum, plasma and urine. It provides useful improvements for b-cell function testing and for evaluating the clinical status of a patient in combination with other diabetes markers. Clin Chem Lab Med 2009;47:834-41.

show abstract

The role of assembly in insulin's biosynthesis

Cited by 481 publications

References 19 publications

Protective hinge in insulin opens to enable its receptor engagement

Protective hinge in insulin opens to enable its receptor engagement

Engineering-enhanced Protein Secretory Expression in Yeast with Application to Insulin

Diagnostic performance of the ARCHITECT C-Peptide immunoassay

Contact Info

Product

Resources

About