Abstract
Background:Dysregulated angiogenesis is a common feature of cardiovascular disease and cancer. Oxidative stress and redox signalling play a crucial role in the control of angiogenesis. At the molecular level this regulation occurs through cysteine thiol modifications on proteins which has the propensity to alter their physiochemical properties. A key redox modification regulating protein function is S-glutathionylation. This reversible modification is enzymatically removed by Glutaredoxin1 (GLRX1). Previous in vivo work highlighted a role for GLRX1 in neovascularisation and angiogenesis. However, the full range of redox-sensitive proteins regulating angiogenesis is not fully understood. This study aimed to uncover redox-sensitive proteins involved in angiogenesis and establish a set of candidate proteins for downstream investigation. Following bioinformatic analysis of candidates, Jagged1 (JAG1) was selected as an interesting and novel target for further investigation. JAG1 is a transmembrane protein that regulates vascular sprouting through cell-cell interactions. Additionally, proteolytic cleavage of JAG1 produces an intracellular (ICD) fragment which regulates transcription.
Methods:
Human cardiac microvascular ECs overexpressing GLRX1 were treated with VEGF165 or hypoxia, stimulating an angiogenic response. Cysteine-reactive tandem mass tags (iodoTMT) were used to label reversibly modified proteins before mass spectrometry. This enabled identification and quantification of redox-sensitive proteins. Bioinformatic analysis of these proteins identified targets involved in angiogenesis, including JAG1. Redox-insensitive variants of JAG1 were generated to establish the function of its redox modifications. Including a ‘redox dead’ (RD) variant containing cysteine to serine substitutions at all seven redox-sensitive cysteines found on JAG1. Redox variants of JAG1 were transiently expressed in ECs and HEK293 cells to determine the effect on JAG1-related processes. DIA proteomics was also performed on the RD variant to characterise protein expression changes.
Results:
IodoTMT proteomics successfully identified redox-sensitive proteins involved in angiogenesis with novel redox modifications, including JAG1, COL4A2, CTGF, FN1, and THBS1. Investigation of the RD JAG1 variant revealed impaired migration in arterial ECs and improved proliferation in HEK293 cells compared to WT JAG1. DIA proteomics identified changes in cell cycle, adhesion and extracellular matrix (ECM) protein levels in HUVECs expressing WT or RD JAG1. Treatment with H2O2 increased formation of the JAG1 ICD fragment in WT but not RD expressing cells. This increase was observed within the nucleus where JAG1 ICD transcriptional control occurs.
Conclusion:
This study showed for the first time that JAG1 undergoes redox modifications, controlling the formation of its ICD signalling fragment. Proteomic analysis demonstrated roles for this fragment in cell cycle, adhesion and ECM protein expression within ECs. JAG1 redox modifications were also shown to have a functional impact on cell migration and proliferation. Additionally, iodoTMT proteomics identified redox-sensitive ECM proteins involved in angiogenesis. Thereby, improving understanding of the redox systems governing this complex biological process.
| Date of Award | 2024 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Sponsors | Medical Research Council |
| Supervisor | Colin Murdoch (Supervisor) & James Cantley (Supervisor) |