Background: The nuclear factor-κB (NF-κB) signalling pathway is a central mediator of inflammatory and immune responses and has been linked to several metabolic and inflammatory diseases, including atherosclerosis. IKKγ/NEMO is essential for NF-κB activation, and its dysfunction in humans is associated with immunodeficiency and inflammation. Atherosclerosis is initiated by vascular injury in response to atherogenic risk factors such as dyslipidemia and hypertension. These factors induce endothelial cell activation, partly through the upregulation of adhesion molecules via NF-κB. In a previous report, we identified NEMO localizing to multivesicular bodies (MVBs). Here, we investigate the membrane trafficking of NEMO in response to NF-κB activation.
Results: Mass spectrometric analyses of NEMO immunoprecipitates suggested vesicle-associated membrane protein 3 (VAMP3), a component of the endosomal trafficking machinery, as an interaction partner. This interaction was confirmed by co-immunoprecipitation and FLIM-FRET analysis in macrophages. Since K63-linked, proteasomal-independent polyubiquitination of NEMO is critical for modulating NF-κB activity, we co-transfected NEMO and VAMP3 with different ubiquitin plasmids. We observed a strong downregulation of NEMO, suggesting a role for VAMP3 and ubiquitin in NF-κB signalling through the direct modulation of NEMO stability. Furthermore, mutation of the VAMP3 ubiquitylation target lysines (K66/68R, K77R) abrogated the formation of enlarged NEMO endosomes and led to a complete block of NF-κB activation. The perinuclear co-localisation of NEMO and VAMP3 suggested a potential role for VAMP3 in the nucleocytoplasmic shuttling of NEMO. To investigate this, we generated a VAMP-SUMO fusion construct. Surprisingly, upon co-transfection and cell fractionation, NEMO was found predominantly in the cytoplasm, indicating an additional role for VAMP3 in regulating NEMO and NF-κB signalling. Analysis of atherosclerotic tissue specimens by immunohistochemistry revealed co-localized expression of VAMP3 and NEMO, suggesting in vivo relevance.
Conclusion: VAMP3 directly interacts with NEMO and modulates NF-κB function in vitro, with probable relevance in vivo
