PXD032784-1

PXD032784 is an original dataset announced via ProteomeXchange.

Title	Insights into the mechanism of human deiodinase 1
Description	The three isoenzymes of iodothyronine deiodinases (DIO1-3) are membrane-anchored homo-dimeric selenoproteins which share the thioredoxin fold structure. Several questions regarding their catalytic mechanisms still remain open. Here, we addressed the roles of several cysteines which are conserved among deiodinase isoenzymes and asked whether they may contribute to dimerization and reduction of the oxidized enzyme with physiological reductants. We also asked whether amino acids previously identified in DIO3 play the same role in DIO1. Human DIO1 and 2 were recombinantly expressed in insect cells with selenocysteine replaced with cysteine (DIO1U126C), or in COS7 cells as selenoprotein. Enzyme activities were studied by radioactive deiodination assays with physiological reducing agents and recombinant proteins were characterized by mass-spectrometry. Mutation of Cys124 in DIO1 prevented reduction by glutathione, while 20 mM dithiothreitol still regenerated the enzyme. Protein thiol reductants, thioredoxin and glutaredoxin, did not reduce DIO1U126C. Mass-spectrometry demonstrated the formation of an intracellular disulfide between the side-chains of Cys124 and Cys(Sec)126. We conclude that the proximal Cys124 forms a selenenyl-sulfide with the catalytic Sec126 during catalysis, which is the substrate of the physiological reductant glutathione. Mutagenesis studies support the idea of a proton-relay pathway from solvent to substrate that is shared between DIO1 and DIO3.
HostingRepository	PRIDE
AnnounceDate	2022-05-31
AnnouncementXML	Submission_2022-05-31_07:35:07.157.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD032784
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Marc Sylvester
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606; scientific name: Spodoptera frugiperda; NCBI TaxID: 7108;
ModificationList	disulfide crosslinked residues
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2022-03-24 13:23:44	ID requested
⏵ 1	2022-05-31 07:35:08	announced

Rodriguez-Ruiz A, Braun D, Pflug S, Brol A, Sylvester M, Steegborn C, Schweizer U, Insights into the Mechanism of Human Deiodinase 1. Int J Mol Sci, 23(10):(2022) [pubmed]

submitter keyword: selenoprotein

selenocysteine

thyroid hormone

kinetic analysis, LC-MSMS

Ulrich Schweizer
contact affiliation	Institut für Biochemie und Molekularbiologie, Universitätsklinikum Bonn, Rheinische Friedrich-Wilhelms- Universität Bonn, Germany
contact email	uschweiz@uni-bonn.de
lab head
Marc Sylvester
contact affiliation	Institute of Biochemistry and Molecular Biology, Medical Faculty, University of Bonn
contact email	msylvest@uni-bonn.de
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD032784
     1. Label: PRIDE project
     2. Name: Insights into the mechanism of human deiodinase 1