Mechanistic insights into the subversion of the linear ubiquitin chain assembly complex by the E3 ligase IpaH1.4/2.5 of Shigella flexneri [IMAGE: PAN LIFENG]

Ubiquitination is a reversible post-translational protein modification that can be regulated by a variety of signal factors and is widely involved in almost every important cellular process. The linear ubiquitin chain assembly complex (LUBAC), which consists of a catalytic subunit HOIP and the two regulatory subunits HOIL-1L and SHARPIN, is the only currently identified E3 ligase complex capable of catalyzing linear poly-Ub chains on protein substrates. LUBAC can be recruited into multiple upstream and downstream signaling pathways to regulate their transduction. The linear poly-Ub chains generated by LUBAC can act as scaffolds to recruit effector proteins, thereby widely participating in NF-κB and other signaling pathways, and play pivotal roles in innate immunity, cell death and antibacterial selective autophagy (xenophagy). Shigella flexneri, a gram-negative bacterium, is the major culprit in bacterial shigellosis, and causes many deaths annually. To evade the host immune response during infection, Shigella flexneri secretes two highly similar E3 ligases, IpaH1.4 and IpaH2.5, to subvert the linear ubiquitin chain assembly complex (LUBAC) of host cells. However, the detailed molecular mechanism underpinning the subversion of LUBAC by IpaH1.4/2.5 remains elusive.

Recently, a research paper titled “Mechanistic insights into the subversion of the linear ubiquitin chain assembly complex by the E3 ligase IpaH1.4 of Shigella flexneri was published in the PNAS journal by Professor Pan Lifeng’s group from the Shanghai Institute of Organic Chemistry (SIOC) of the Chinese Academy of Sciences (CAS). In this paper, using various biochemical techniques, such as Nuclear Magnetic Resonance (NMR), Isothermal Titration Calorimetry (ITC), and Fast Protein Liquid Chromatography (FPLC), they discovered that IpaH1.4 specifically recognizes HOIP and HOIL-1L through its LRR domain by binding to the HOIP RING1 domain and the HOIL-1L UBL domain, respectively. They determined the crystal structures of the IpaH1.4 LRR/HOIP RING1 complex, the IpaH1.4 LRR/HOIL-1L UBL complex and the HOIP RING1/UBE2L3 complex for the first time, elucidated the binding mechanisms of IpaH1.4 with HOIP and HOIL-1L, and unveiled that the recognition of HOIP by IpaH1.4 can inhibit the E2-binding of HOIP. Additionally, they demonstrated that the interaction of IpaH1.4 LRR with HOIP RING1 or HOIL-1L UBL is essential for the ubiquitination of HOIP or HOIL-1L in vitro as well as the suppression of NF-κB activation by IpaH1.4 in cells. This work elucidates the three different strategies simultaneously adopted by IpaH1.4 to disrupt the function of LUBAC, thereby inhibiting the activation of the NF-κB signaling pathway in host cells and antibacterial selective autophagy. The strategies are to mediate the K48-linked ubiquitination of HOIP for proteasomal degradation, to inhibit the E3 activity of LUBAC by blocking the E2-binding of HOIP, and to suppress the capacity of HOIL-1L binding to HOIP and disturb the stability of LUBAC by acting on the UBL domain of HOIL-1L and mediating K48-linked ubiquitination and subsequent proteasomal degradation of HOIL-1L.

In summary, this study reveals, for the first time, the molecular mechanism underlying the multiple tactics adopted by the E3 ligase IpaH1.4 of S. flexneri to hijack the LUBAC complex of host cells from a biochemical and structural perspective, and provided a new potential target for drug development against S. flexneri infection in the future.

The paper’s associate researcher is Dr. Liu Jianping, while PhD student Wang Yaru from Professor Pan’s group and PhD student Wang Danni from Professor Yao Yufeng’s group are its co-authors.

For more information, please contact:

Professor Pan Lifeng

E-mail: panlf@sioc.ac.cn

Shanghai Institute of Organic Chemistry (SIOC),

Chinese Academy of Sciences

Source: Shanghai Institute of Organic Chemistry (SIOC),

Chinese Academy of Sciences