University of Oulu

Novel regulatory mechanisms and structural aspects of oxidative protein folding

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Author: Moilanen, Antti1,2
Organizations: 1University of Oulu Graduate School
2University of Oulu, Faculty of Biochemistry and Molecular Medicine
Format: ebook
Version: published version
Access: open
Online Access: PDF Full Text (PDF, 3.3 MB)
Persistent link: http://urn.fi/urn:isbn:9789526228471
Language: English
Published: Oulu : University of Oulu, 2021
Publish Date: 2021-02-08
Thesis type: Doctoral Dissertation
Defence Note: Academic dissertation to be presented with the assent of the Doctoral Training Committee of Health and Biosciences of the University of Oulu for public defence in the Leena Palotie auditorium (101A) of the Faculty of Medicine (Aapistie 5 A), on 15 February 2021, at 12 noon
Tutor: Professor Lloyd Ruddock
Reviewer: Professor Carolyn Sevier
Doctor Adam Benham
Opponent: Professor Neil Bulleid
Description:

Abstract

Efficient maturation of disulfide-containing proteins in the endoplasmic reticulum (ER) requires facilitated folding, aided by various folding catalysts and chaperones. As protein folding is a complex process, proteins may, even in the presence of endogenous folding factors, form misfolded states. Therefore, the cell has acquired several layers of quality control mechanisms to keep the folding load in the ER in balance with the folding capacity. The activity of folding catalysts is regulated to avoid stress connected to hyperoxidation. Other quality control mechanisms include the unfolded protein response (UPR) and ER-associated protein degradation (ERAD). The UPR consists of signaling cascades that sense unfolded protein stress and upregulate folding factors and other components to relieve the stress. ERAD directly removes non-native proteins.

The main folding catalysts that introduce disulfide bonds to folding proteins in the ER are members of the ER oxidoreductin 1 (Ero1) family and protein disulfide isomerases (PDI). Ero1 is a sulfhydryl oxidase that reduces molecular oxygen to hydrogen peroxide to form disulfides. The newly generated disulfides are then transferred during the catalytic cycle to PDI which catalyzes disulfide formation in folding proteins. The Ero1-PDI pathway has been studied extensively by cell-based techniques, and its activity is regulated in a redox dependent manner by rearranging regulatory disulfides. However, the kinetic aspects of these regulatory functions have remained poorly characterized.

In this study, we report comprehensive kinetic measurements for the Ero1-PDI pathway that reveal novel regulatory mechanisms for oxidative protein folding (OPF). These include a mechanism consisting of high affinity and apparent cooperativity for substrate oxygen to allow Ero1 to function efficiently at hypoxic conditions. We also show that different human Ero1 isoforms have differential dependence for substrate PDI with one of the isoforms, Ero1α, showing strong dependence for PDI for both redox activation as well as the catalytic cycle. This observation allowed us to describe a potential crosstalk between OPF and ERAD, in which OPF by the Ero1α-PDI pathway is downregulated, to potentially increase the efficiency of ERAD which requires reduced protein substrates. This work is concluded by a structural study of a novel chaperone, pERp1, that is crucial for our immunity, as it is involved in the folding of some immunoglobulins.

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Tiivistelmä

Disulfidisidoksia sisältävien proteiinien laskostumista endoplasmakalvostossa avustavat erilaiset laskostumiskatalyytit ja kaperonit. Koska laskostuminen on monimutkaista, voivat proteiinit laskostumistekijöistä huolimatta laskostua väärin. Endoplasmakalvostoon onkin kehittynyt useita laadunvarmistusmekanismeja, jotka pitävät laskostumistaakan ja -kapasiteetin tasapainossa. Laskostumiskatalyyttien aktiivisuutta voidaan säädellä, minkä avulla vältetään ylihapettumiseen liittyvä stressi. Muihin laadunvarmistuskeinoihin kuuluvat UPR (unfolded protein response) ja ERAD (endoplasmic reticulum-associated degradation). UPR tunnistaa laskostumattomien proteiinien aiheuttaman stressin ja saa aikaan mm. laskostumistekijöiden lisätuotantoa stressin vähentämiseksi. ERAD poistaa suoraan laskostumattomia proteiineja hajottamalla niitä.

Ero1- ja PDI-proteiiniperheiden jäsenet toimivat pääasiallisina laskostumiskatalyytteinä, jotka muodostavat disulfideja laskostuviin proteiineihin. Ero1 on sulfhydryylioksidaasi, joka pelkistää hapen veryperoksidiksi muodostaessaan disulfidin. Uuden disulfidin Ero1 siirtää sitten PDI:lle, joka puolestaan katalysoi disulfidien muodostuksen laskostuvissa proteiineissa. Ero1-PDI-systeemiä on tutkittu paljon solutasolla, ja sen aktiivisuutta voidaan säädellä järjestelemällä sen omia säätelydisulfideja. Tämän säätelyjärjestelmän entsyymikinetiikka tunnetaan kuitenkin huonosti.

Tässä työssä esittelemme Ero1-PDI-systeemin laajat entsyymikinetiikan mittaukset, jotka paljastavat uusia säätelymenetelmiä proteiinien laskostumiseen. Näihin menetelmiin kuuluvat mm. tehokkuus hypoksisissa olosuhteissa ja erilaiset kineettiset ominaisuudet ihmisen Ero1-perheen jäsenten välillä. Tulokset osoittavat, että Ero1α vaatii korkeat pitoisuudet PDI-substraattia sekä aktivoituakseen että disulfidien siirtoon PDI:lle. Näiden tulosten perusteella havaitsimme mahdollisen laskostumisen ja ERAD:n välisen vuorovaikutuksen, jossa laskostumisen tehokkuutta lasketaan ERAD:n toiminnan tehostamiseksi. Lisäksi esittelemme kiderakenteen uudelle kaperonille, pERp1:lle, joka vasta-aineiden laskostumistekijänä on tärkeä immuniteetillemme.

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Osajulkaisut / Original papers

Osajulkaisut eivät sisälly väitöskirjan elektroniseen versioon / Original papers are not included in the electronic version of the dissertation.

  1. Moilanen, A., Korhonen, K., Saaranen, M. J., & Ruddock, L. W. (2018). Molecular analysis of human Ero1 reveals novel regulatory mechanisms for oxidative protein folding. Life Science Alliance, 1(3), e201800090. https://doi.org/10.26508/lsa.201800090

    Rinnakkaistallennettu versio / Self-archived version

  2. Moilanen, A., & Ruddock, L. W. (2020). Non-native proteins inhibit the ER oxidoreductin 1 (Ero1)–protein disulfide-isomerase relay when protein folding capacity is exceeded. Journal of Biological Chemistry, 295(26), 8647–8655. https://doi.org/10.1074/jbc.ra119.011766

    Rinnakkaistallennettu versio / Self-archived version

  3. Sowa, S. T., Moilanen, A., Biterova, E., Saaranen, M. J., Lehtiö, L., & Ruddock L. W. (2020). High-resolution crystal structure of human pERp1, a saposin-like protein crucial for IgA, IgM and integrin maturation in the endoplasmic reticulum. Manuscript submitted for publication. https://doi.org/10.1016/j.jmb.2021.166826

    Rinnakkaistallennettu versio / Self-archived version

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Series: Acta Universitatis Ouluensis. A, Scientiae rerum naturalium
ISSN: 0355-3191
ISSN-E: 1796-220X
ISSN-L: 0355-3191
ISBN: 978-952-62-2847-1
ISBN Print: 978-952-62-2846-4
Issue: 754
Type of Publication: G5 Doctoral dissertation (articles)
Field of Science: 1182 Biochemistry, cell and molecular biology
Subjects:
PDI
UPR
Copyright information: © University of Oulu, 2021. This publication is copyrighted. You may download, display and print it for your own personal use. Commercial use is prohibited.