br Materials and methods br Results
Materials and methods
Discussion Innate immunity plays an essential role in the control and elimination of bacterial infection through production of inflammatory cytokines (Liu et al., 2015). Production of inflammatory cytokines requires the recognition of bacterial pathogen-associated molecular patterns by TLRs receptors. Full activation of TLR4 could be significant part in the initiation of innate response and subsequent elimination of invading pathogens (O’Neill and Bowie, 2007).It has been well-documented that TLR4 could be activated through either MyD88-dependent or TRIF-dependent pathways. TLR4-deficient mice show a suppressed production of type I IFNs and proinflammatory cytokines and are highly susceptible to bacterial infection (Deng et al., 2013). So, recently, much attention has been devoted to identifying the regulators of TLR4-triggered immune response attracts. Our group previously reported that CHIP could positively regulate TLR4 signaling by recruiting and polyubiquitinating Src and atypical PKC?? (Yang et al., 2011). In this study, we provide evidence for the critical role of TRIM7 in TLR4-associated signaling pathways and the production of TLR4-induced inflammatory cytokine. Overexpression of TRIM7 results in the activation of MAPKs, NF-κB and IRF3, and leads to an increase in LPS-triggered production of type I IFN, TNF-α and IL-6. TRIM7 truncation analysis reveals that deletion of the E3 ligase domain may significantly attenuate the effect of TRIM7 on LPS-triggered innate response. These results indicate that TRIM7 may positively regulate the TLR4-mediated signaling via its E3 ligase domain. To our knowledge, this is the first report showing the biological significance of E3 ligaseon TRIM7 in the regulation of the innate inflammatory response. TRIM7 has been reportedly involved in some important biological processes including tumor cell proliferation and glycogen metabolism (Montori-Grau et al., 2018; Zhan et al., 2015). Here, we show that TRIM7 is critical for the TLR4-associated signaling pathways and the production of TLR4-induced inflammatory cytokine. However, interestingly, LPS treatment may reduce TRIM7 expression initially in macrophages. Considering the importance of TLR4 in the host anti-bacterial immunity, one possibility for this result is that bacteria may modulate TRIM7 expression to escape immune surveillance and facilitate bacteria replication. Moreover, we demonstrate that TRIM7 is primarily expressed in antigen-presenting CC-223 including macrophages, dendritic cells and B cells. Over-expression and silencing experiments above indicate that TRIM7 could facilitate the LPS-initiated TLR4 signaling by promoting the phosphorylation levels of MAPKs, NF-κB and IRF3. TRIM7 has been functionally linked to lys63-ubiquitination-mediated protein activation. However, the role of TRIM7 in innate immunity and inflammation is still limited. As previously reported, TRIM7 as an E3 ligase could mediate K63-linked ubiquitination of the AP-1 coactivator RACO-1, then leading to RACO-1 protein stabilization (Chakraborty et al., 2015).Although in the present study no protein is shown to interact with TRIM7 or be modified with K63-ubiquitination, we have already prepared different truncated TRIM7 including delta RING (82–510 aa), N165 (1–165 aa), N275 (1–275 aa), C165 (166–510 aa), C275 (276–510 aa)(data not shown), which will be used to further investigate how TRIM7 functions in the TLR4-mediated innate response.
Conflicts of interest
Introduction Targeting proteins for degradation by hijacking the ubiquitin-proteasome system with small molecules is a powerful modality of intervention into biology, and an emerging therapeutic strategy.1, 2, 3, 4 A primary approach to targeted protein degradation involves the design of PROTACs (PROteolysis-Targeting Chimeras). PROTACs are bifunctional compounds that form a ternary complex with a target protein of interest and an E3 ubiquitin ligase, such that the target protein is ubiquitinated by the hijacked E3 ligase and subsequently degraded by the proteasome.5, 6 PROTACs are defined by a catalytic, sub-stoichiometric mode of action that can allow for rapid, profound and selective target depletion inside cells, and an extended duration of action, also in vivo.7, 8, 9, 10 Because their mode of action differs from that of conventional inhibitors, the concentrations at which PROTACs exert degradation activity are often much lower than expected based on their dissociation constants with the target protein.11, 12, 13, 14 Furthermore, PROTAC’s selectivity can be greater than the binding selectivity of the ligands alone, allowing to discriminate between highly similar proteins or isoforms in ways that are not possible with occupancy-based inhibitors.8, 11, 12, 15, 16, 17 Within the past four years, potent and selective PROTACs have been designed to hijack either the von Hippel-Lindau (VHL) or cereblon (CRBN) E3 ligase against a target protein of interest.18, 19 Targets that have been shown to be degraded by PROTACs include members of bromodomain-containing proteins such as the BET proteins (Brd2, Brd3 and Brd4),7, 8, 9, 14, 15, 17, 20, 21 amongst other epigenetic protein classes;22, 23, 24, 25, 26 protein kinases;10, 12, 27, 28, 29, 30, 31 as well as non-bromodomain and non-kinase target proteins.32, 33, 34, 35 Recent progress in understanding principles of PROTAC mode of action, and demonstration of applicability across different target classes, suggest that PROTACs have the potential to target new protein families, including proteins that are difficult to block using current approaches. Clinical validation of small molecules inducing protein degradation is provided by recent discoveries on the molecular mechanism of thalidomide and related clinical anticancer immunomodulatory drugs (IMiDs) such as lenalidomide and pomalidomide, which induce the proteasomal-dependent degradation of cancer-driving proteins.36, 37 More recently, a PROTAC compound (ARV-110) that targets the androgen receptor for degradation has been announced as a clinical candidate.