CRISPR/Cas9-mediated modification of the extreme C-terminus impairs PDGF-stimulated activity of Duox2

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Duox2 is an NADPH oxidase enzyme family member involved in various cellular processes.
• Duox2 and hydrogen peroxide (H2O2) are known to play a role in platelet-derived growth factor (PDGF)-induced fibroblast migration.

Purpose of the Study:

• To investigate the role of the C-terminal region of Duox2 in PDGF-stimulated activity.
• To determine if the extreme C-terminus is required for Duox2 function and H2O2 production.

Main Methods:

• CRISPR/Cas9-mediated genome editing was used to modify Duox2.
• Fibroblast cell lines were engineered to co-express wild-type or C-terminally modified Duox2 with a fluorescent H2O2 probe (Hyper).

Main Results:

• Nonsense substitution of the final 23 amino acids of Duox2 abolished PDGF-stimulated intracellular H2O2 production.
• These C-terminal modifications also resulted in a complete loss of PDGF-stimulated fibroblast migration.
• Duox2 expression levels and those of other NADPH oxidases remained unaffected by the mutations.

Conclusions:

• The extreme C-terminus of Duox2 is indispensable for its functional activity in response to PDGF.
• The conserved nature of this C-terminal region suggests a potential role in the activity of other NADPH oxidases.