Design and Development of a Novel Immunological Method to Quantify target-specific cysteine redox state in a microplate

Reactive oxygen species (ROS) regulate numerous fundamental processes such<br/>as innate immune response, commonly by modifying the thiol groups (RSH) of<br/>functionally relevant cysteine (Cys) residues of signalling proteins. The ability to<br/>measure target-specific protein thiol oxidation underpins our capability to<br/>interrogate these processes, which is an ongoing bottleneck, as the currently<br/>available methods face various limitations. This was showcased in the present<br/>study where the redox state of interleukin 1 receptor associated kinase (IRAK) 1<br/>could not be measured using mobility shift assays. To address this technical<br/>impediment, a novel technique called RedoxiFluor was developed. RedoxiFluor<br/>involves the labelling of reduced, and reversibly oxidised thiols with spectrally<br/>distinct fluorescent probes, which are used to quantify thiol oxidation in<br/>percentage terms. Proof-of-principle studies were used to validate RedoxiFluor,<br/>where the redox state of the bulk thiol pool could be accurately quantified, when<br/>applied to crude lysates. To achieve target-specificity, antibody-functionalised<br/>microplates were utilised, where IRAK-1-specific thiol oxidation could be<br/>accurately quantified in percentage terms. Using a pair-matched detector<br/>antibody enabled target protein concentration to be quantified in tandem with<br/>percentage oxidation, where combining the two enabled molar quantification of<br/>target-specific thiol oxidation. Overall, RedoxiFluor is currently the only assay<br/>that can measure target-specific redox state in relation to the bulk thiol pool, in<br/>percentages and moles, in a microplate. Next, lipopolysaccharide (LPS) -induced<br/>thiol oxidation of several proteins in THP-1 cells was investigated using<br/>RedoxiFluor. LPS led to an increase in IRAK-1-specific thiol oxidation, which was<br/>accompanied with an increase in protein tyrosine (Tyr) phosphatase (PTP) 1B<br/>(PTP1B), Src homology 2 domain-containing protein Tyr phosphatase 1 (SHP-<br/>1), cluster of differentiation 45 (CD45) and the cα subunit of protein phosphatase<br/>2A (PP2A) -specific thiol oxidation, without altering the thiol oxidation of Src<br/>homology 2 domain-containing protein Tyr phosphatase 2 (SHP-2), phosphatase<br/>and tensin homolog (PTEN), cγ subunit of protein phosphatase 2B (PP2B) and<br/>the bulk thiol pool. These findings provide further insight into the redox regulation<br/>of endotoxin signalling and showcase the various advantages of RedoxiFluor.

Name dc:type.qualificationname

Doctor of Philosophy (awarded by UHI)

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

University of the Highlands and Islands

Year dc:date.issued

2023

Author dc:creator

• Tuncay, Ahmet

Language dc:language

eng

Identifier

oai:pure.atira.dk:studenttheses/6fac8e0f-baed-4877-bacc-44a9a8c50a6f

OAI identifier oai:identifier

oai:pure.atira.dk:studenttheses/6fac8e0f-baed-4877-bacc-44a9a8c50a6f