Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies

Freja Bohr; Søren S.R. Bohr; Narendra Kumar Mishra; Nicolás Sebastian González-Foutel; Henrik Dahl Pinholt; Shunliang Wu; Emilie Milan Nielsen; Min Zhang; Magnus Kjaergaard; Knud J. Jensen; Nikos S. Hatzakis

doi:10.1038/s42003-022-04386-6

Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies

Freja Bohr, Søren S.R. Bohr, Narendra Kumar Mishra, Nicolás Sebastian González-Foutel, Henrik Dahl Pinholt, Shunliang Wu, Emilie Milan Nielsen, Min Zhang, Magnus Kjaergaard, Knud J. Jensen^*, Nikos S. Hatzakis

^*Corresponding author af dette arbejde

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review

5 Citationer (Scopus)

Abstract

Insulin formulations with diverse oligomerization states are the hallmark of interventions for the treatment of diabetes. Here using single-molecule recordings we firstly reveal that insulin oligomerization can operate via monomeric additions and secondly quantify the existence, abundance and kinetic characterization of diverse insulin assembly and disassembly pathways involving addition of monomeric, dimeric or tetrameric insulin species. We propose and experimentally validate a model where the insulin self-assembly pathway is rerouted, favoring monomeric or oligomeric assembly, by solution concentration, additives and formulations. Combining our practically complete kinetic characterization with rate simulations, we calculate the abundance of each oligomeric species from nM to mM offering mechanistic insights and the relative abundance of all oligomeric forms at concentrations relevant both for secreted and administrated insulin. These reveal a high abundance of all oligomers and a significant fraction of hexamer resulting in practically halved bioavailable monomer concentration. In addition to providing fundamental new insights, the results and toolbox presented here can be universally applied, contributing to the development of optimal insulin formulations and the deciphering of oligomerization mechanisms for additional proteins.

Originalsprog	Engelsk
Artikelnummer	178
Tidsskrift	Communications Biology
Vol/bind	6
Nummer	1
ISSN	2399-3642
DOI	https://doi.org/10.1038/s42003-022-04386-6
Status	Udgivet - dec. 2023

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10.1038/s42003-022-04386-6

Citationsformater

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title = "Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies",

abstract = "Insulin formulations with diverse oligomerization states are the hallmark of interventions for the treatment of diabetes. Here using single-molecule recordings we firstly reveal that insulin oligomerization can operate via monomeric additions and secondly quantify the existence, abundance and kinetic characterization of diverse insulin assembly and disassembly pathways involving addition of monomeric, dimeric or tetrameric insulin species. We propose and experimentally validate a model where the insulin self-assembly pathway is rerouted, favoring monomeric or oligomeric assembly, by solution concentration, additives and formulations. Combining our practically complete kinetic characterization with rate simulations, we calculate the abundance of each oligomeric species from nM to mM offering mechanistic insights and the relative abundance of all oligomeric forms at concentrations relevant both for secreted and administrated insulin. These reveal a high abundance of all oligomers and a significant fraction of hexamer resulting in practically halved bioavailable monomer concentration. In addition to providing fundamental new insights, the results and toolbox presented here can be universally applied, contributing to the development of optimal insulin formulations and the deciphering of oligomerization mechanisms for additional proteins.",

author = "Freja Bohr and Bohr, {S{\o}ren S.R.} and Mishra, {Narendra Kumar} and Gonz{\'a}lez-Foutel, {Nicol{\'a}s Sebastian} and Pinholt, {Henrik Dahl} and Shunliang Wu and Nielsen, {Emilie Milan} and Min Zhang and Magnus Kjaergaard and Jensen, {Knud J.} and Hatzakis, {Nikos S.}",

note = "Publisher Copyright: {\textcopyright} 2023, The Author(s).",

year = "2023",

month = dec,

doi = "10.1038/s42003-022-04386-6",

language = "English",

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T1 - Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies

AU - Bohr, Freja

AU - Bohr, Søren S.R.

AU - Mishra, Narendra Kumar

AU - González-Foutel, Nicolás Sebastian

AU - Pinholt, Henrik Dahl

AU - Wu, Shunliang

AU - Nielsen, Emilie Milan

AU - Zhang, Min

AU - Kjaergaard, Magnus

AU - Jensen, Knud J.

AU - Hatzakis, Nikos S.

PY - 2023/12

Y1 - 2023/12

N2 - Insulin formulations with diverse oligomerization states are the hallmark of interventions for the treatment of diabetes. Here using single-molecule recordings we firstly reveal that insulin oligomerization can operate via monomeric additions and secondly quantify the existence, abundance and kinetic characterization of diverse insulin assembly and disassembly pathways involving addition of monomeric, dimeric or tetrameric insulin species. We propose and experimentally validate a model where the insulin self-assembly pathway is rerouted, favoring monomeric or oligomeric assembly, by solution concentration, additives and formulations. Combining our practically complete kinetic characterization with rate simulations, we calculate the abundance of each oligomeric species from nM to mM offering mechanistic insights and the relative abundance of all oligomeric forms at concentrations relevant both for secreted and administrated insulin. These reveal a high abundance of all oligomers and a significant fraction of hexamer resulting in practically halved bioavailable monomer concentration. In addition to providing fundamental new insights, the results and toolbox presented here can be universally applied, contributing to the development of optimal insulin formulations and the deciphering of oligomerization mechanisms for additional proteins.

AB - Insulin formulations with diverse oligomerization states are the hallmark of interventions for the treatment of diabetes. Here using single-molecule recordings we firstly reveal that insulin oligomerization can operate via monomeric additions and secondly quantify the existence, abundance and kinetic characterization of diverse insulin assembly and disassembly pathways involving addition of monomeric, dimeric or tetrameric insulin species. We propose and experimentally validate a model where the insulin self-assembly pathway is rerouted, favoring monomeric or oligomeric assembly, by solution concentration, additives and formulations. Combining our practically complete kinetic characterization with rate simulations, we calculate the abundance of each oligomeric species from nM to mM offering mechanistic insights and the relative abundance of all oligomeric forms at concentrations relevant both for secreted and administrated insulin. These reveal a high abundance of all oligomers and a significant fraction of hexamer resulting in practically halved bioavailable monomer concentration. In addition to providing fundamental new insights, the results and toolbox presented here can be universally applied, contributing to the development of optimal insulin formulations and the deciphering of oligomerization mechanisms for additional proteins.

U2 - 10.1038/s42003-022-04386-6

DO - 10.1038/s42003-022-04386-6

M3 - Journal article

C2 - 36792809

AN - SCOPUS:85148114502

SN - 2399-3642

VL - 6

JO - Communications Biology

JF - Communications Biology

IS - 1

M1 - 178

ER -

Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies

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