IKKγ/NEMO recycling endosome trafficking pathway is regulated via VAMP3 ubiquitylation

Background: The nuclear factor-κB (NF-κB) signalling pathway has been linked to several metabolic and inflammatory diseases such as atherosclerosis. IKKγ/NEMO is essential for NF-κB activation, Atherosclerosis is initiated by vascular injury as a response to atherogenic risk factors such as dyslipidemia, diabetes, hypertension, and cigarette smoking. These risk factors induce endothelial cell activation through upregulation of adhesion molecules, particularly via NF-κB. The NF-κB pathway is a central signalling pathway for inflammatory and immune responses. IKKγ/NEMO is essential for NF-κB activation, and NEMO dysfunction in humans has been linked to immunodeficiency and inflammation. In a previous report, we identified NEMO localization into multivesicular bodies (MVBs). Here, we addressed the question of the membrane traffic of NEMO in response to NF-kB activation.

Results: Mass spectrometric analyses of NEMO immune-precipitates suggested that vesicle associated membrane protein (VAMP3), a component of the endosomal trafficking machinery, as a interaction partner of NEMO by immune-precipitation of VAMP3 and subsequent analysis of the immune complexes for the presence of NEMO or VAMP3. The results of the immune-precipitation were confirmed by FRET and FLIM-FRET analysis in macrophages. It is well known that K63-linked, proteasomal-independent polyubiquitination of NEMO is critical for modulation of NF-κB activity. Therefore, we performed co-transfection of NEMO and VAMP3 with different ubiquitin plasmids. We observed strong downregulation of NEMO by co-transfection with VAMP3 and different forms of ubiquitin. This observation suggests a role of VAMP3 and ubiquitins in NF-κB signalling through direct modulation of NEMO stability. To further investigate the significance of VAMP3 for NF-κB activation, we transfected cells with siRNAs targeting VAMP3. Indeed, reduction of endogenous VAMP3 expression levels resulted in decreased levels of NEMO and consequently reduction of NF-κB activation. 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.

Atherosclerotic tissue specimens were analyzed by immune-histochemistry. Both, VAMP3 and NEMO were found co-localized expressed suggesting in vivo relevance.

Conclusion: VAMP3 directly interacts with NEMO and, this way, modulates NF-κB function in vitro and probably in vivo, as well.