GFP Reporter Cell Line - RAW 264.7

Mycobacterium tuberculosis (Mtb) is the leading infectious pathogen responsible for human mortality, infecting approximately one-quarter of the global population. During infection, Mtb evades the host immune response by secreting effector proteins; these proteins interfere with and modulate host immune reactions, thereby helping the bacteria withstand potent antimicrobial attacks. Among these, the PE5 protein likely plays a pivotal role in the pathogenesis of mycobacteria. Here, researchers employed affinity purification coupled with mass spectrometry (AP-MS) to conduct an in-depth investigation into the molecular functions of PE5, identifying it as an interacting partner of a host E3 ubiquitin ligase complex-the CRL2 complex. PE5 binds to the CRL2 complex via a C-terminal Gly-Gly motif, which is recognized and bound by KLHDC2, a substrate receptor subunit of CRL2. Notably, while PE5 itself does not undergo ubiquitination, it is targeted for degradation upon binding to KLHDC2. Interestingly, the binding of PE5 enhances the autoubiquitination of KLHDC2; however, this autoubiquitination neither impairs KLHDC2's ability to degrade its known substrates nor affects the CRL2 complex's capacity to degrade substrates recognized by other substrate receptors. Thus, despite binding to the CRL2-KLHDC2 complex without undergoing ubiquitination itself, PE5 appears not to impede the overall activity of the CRL2 complex.

Given that PE5 is capable of binding to the substrate-binding domain of KLHDC2, researchers hypothesized that PE5 might be a substrate of the CRL2-KLHDC2 complex. To validate this hypothesis, they first monitored the degradation of these two proteins in lysates from RAW 264.7 cells stably expressing GFP, GFP-PE5, or GFP-PE5ΔGG, utilizing cycloheximide chase assays. The results demonstrated that GFP-PE5ΔGG remained stable over several hours, whereas GFP-PE5 exhibited a half-life of approximately 3 hours and was almost completely degraded after 5 hours (Figure 1A-B). This disparity in stability suggests that PE5's ability to bind to KLHDC2 may regulate its own degradation process. Subsequently, the researchers treated RAW 264.7 cells stably expressing GFP, GFP-PE5, or GFP-PE5ΔGG with the pan-Cullin inhibitor MLN4924 and the proteasome inhibitor MG-132, respectively. The results revealed that, compared to untreated cells, the expression levels of GFP-PE5 were significantly elevated in cells treated with either MLN4924 or MG-132 (Figure 1C-D). This phenomenon was observed exclusively with PE5 and not with PE5ΔGG, which is unable to bind to KLHDC2. This indicates that PE5 is degraded via the proteasomal pathway and that this degradation process is dependent on the activity of CRL2. Furthermore, compared to control cells, the expression levels of PE5 were significantly increased in cells where KLHDC2 had been knocked down (Figure 1E-F). Knocking down KLHDC2 similarly enhanced the stability of PE5 alone, without affecting the stability of PE5ΔGG. This result further confirms that the stability of PE5 is indeed contingent upon its binding to KLHDC2.

Figure 1. PE5 is degraded but not ubiquitinated by CRL2-KLHDC2. (Madduri, Bala TSA, et al., 2025)