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

Abstract

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

Date of Award	31 Jul 2023
Original language	English
Awarding Institution	University of the Highlands and Islands
Sponsors	ESF studentship
Supervisor	James Cobley (Supervisor) & Holger Husi (Supervisor)