Ubiquitinated TRAF2 and BIRC2/3 were reported to recruit an additional E3 ligase complex, the linear ubiquitin (Ub) chain assembly complex (LUBAC). LUBAC is thought to bind K63 chains on BIRC2/3 but produce Met1 (M1)-linked (also known as linear) Ub chains to facilitate recruitment and Met1-linked ubiquitination of NEMO (IKBKG), the regulatory subunit of the IKK complex (Haas TL et al. 2009). LUBAC enhances NEMO interaction with the TNFR1 receptor signaling complex and thus IKK complex, stabilizes this protein complex, and promotes efficient TNF-induced activation of NFkappaB resulting in apoptosis inhibition (Haas TL et al. 2009)

LUBAC consists of the chain-assembling E3 ligase HOIP as well as HOIL-1 and SHARPIN (Kirisako T et al. 2006; Walczak H et al. 2012). Importantly, deletion of the LUBAC component SHARPIN in mice or mutation of HOIL-1 in humans, lead to hyperinflammatory phenotypes, indicating key roles of LUBAC and linear Ub chains in the response to infection and inflammation (Gerlach B et al. 2011; Ikeda F et al. 2011; Tokunaga F et al. 2011; Boisson B et al. 2012).

HOIP belongs to the RING-between-RING (RBR) family of E3 ligases and is the catalytic component of LUBAC (Spratt DE et al. 2014). RBR E3 ligase domain and a conserved C-terminal extension of HOIP are responsible for assembling Met1-linked chains (Smit JJ et al., 2012; Stieglitz B et al. 2012 and 2013). HOIP was reported to act as RING/HECT hybrids, employing RING1 to recognize ubiquitin-loaded E2 while a conserved cysteine in RING2 domain subsequently forms a thioester intermediate with the transferred or “donor” ubiquitin (Stieglitz B et al. 2013). A Ub-associated (UBA) domain mediates interactions with HOIL-1L (Yagi H et al. 2012), while N-terminal PUB domain may be involved in interaction with regulatory proteins such as OTULIN to control NFkappaB signaling (Elliott PR et al. 2014). HOIP also comprises several NPL4 zinc finger (NZF) Ub binding domains (UBDs) that target it to ubiquitinated proteins (Haas et al. 2009; Fujita H et al. 2014).

Structural analysis revealed that NZF1 of HOIP can simultaneously bind both leucine zipper (CoZi) domains of NEMO (IKBKG) and ubiquitin and that both interactions are involved in TNF α-mediated NFkappaB activation (Fujita H et al. 2014). In addition, NEMO (IKBKG) ubiquitination required RBR domain of HOIL-1L (Smit JJ et al. 2013).