Written by | Spring dawn
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In the human body, on average, 5 partners per protein need to assemble together into a multi-subunit complex to play a role in cell information transmission [1], a form of cooperation that resembles a molecular machine. How does this modular complex get the right assembly in the right place at the right time? It is an important puzzle in biology.
A very important presence in these modular complexes is the Cullin-RING E3 ligase family (CRLs). The E3 ligase of the CRL family controls cell division, differentiation, and survival. CRL is an important ubiquitinase that binds a catalytic core built around a Cullin scaffold with approximately 300 exchangeable substrate adaptors (Adaptor). To ensure the smooth and orderly conduct of signal transduction, cells must continuously form new CRLs by pairing substrate-bound artigmachines with Cullin, but how this series of assembly processes occurs is unclear.
On February 3, 2022, Michael Rapé's research team at the University of California, Berkeley, published a research paper titled Co-adaptor driven assembly of a CUL3 E3 ligase complex in the journal Molecular Cell, in which the authors demonstrated that the formation of a single CRL complex is a strictly regulated process. Taking CUL3 KLHL12 as an example, its co-aligning protein PEF1-ALG2 releases KLHL12 through an assembly inhibitor of the endoplasmic reticulum before the modification of a single ubiquitinated substrate, thereby initiating the formation of CRL3. Since co-aligner proteins also help recruit substrates, target recognition is efficiently combined with ubiquitination in CRL assembly. The discovery that modular E3 ligases are critical for signal transduction is complementary to general artignation protein exchange.
CRL3 E3 ligase has a catalytic core consisting of CUL3 and RBX1 and a junction region containing the BTB domain (Figure 1). Michael Rape's research team reported in 2016 that CUL3KLHL12 needs to co-articulate PEF1-ALG2 to stably identify its oligomeric substrate SEC31A[2], thereby regulating the degradation process of endoplasmic reticulum secretory vesicles or autophagosomes. In this new work, the authors found that the co-aligning protein promotes CRL3 assembly differently from its role in recruiting substrates to E3 ligase, and it is the co-aligning protein subunit PEF1 that drives the formation of the CUL3KLHL12 complex.
Figure 1: Structure of crL3 E3 ligase
Mass spectrometry findings suggest that the deletion of PEF1 increases the binding of KLHL12 to SEC31A, while the binding to CUL3 is significantly weakened. At the same time, the deletion of PEF1 promotes the interaction of the endoplasmic omentum protein Lunapark (LNP) with KLHL12, but KLHL12 does not bind to co-aligning proteins or CUL3, which is an inhibitor of CRL3 assembly. Further PEF1 overexpression had the opposite effect, reducing the interaction between KLHL12 and LNP while enriching CUL3.
The team's 2016 report also mentioned that the lack of Ca2+ would prevent the combination of KLHL12 and CUL3[2]. In this new work, the authors found that calcium signaling regulates the competition between co-aligning proteins and LNPs, so it is calcium signaling that may physiologically trigger CUL3KLHL12 activation.
The authors then investigated how PEF1 interacts with CUL3 KLHL12, which requires KLHL12-dependent BTB domains to recruit CUL3, and single ubiquitination of PEF1 stabilizes and activates CUL3 KLHL12 for efficient substrate modification. So who regulates the de-ubiquitination process on the contrary? Once CUL3 KLHL12 lets go of its target, CAND1 releases KLHL12 from CUL3 and USP9X takes ubiquitin from PEF1, inactivating CUL3 KLHL12. The LNP then recaptures KLHL12 before moving on to the next round of assembly (Figure 2).
Figure 2: CUL3 E3 ligase complex assembly.
It can be seen that the co-aligner protein can not only recruit substrates, but also drive the assembly of CRLs. In the process of studying CUL3 KLHL12, the authors found that even though the artigmaking protein KLHL12 has bound to the target, the formation of the E3 ligase still requires its auxiliary artigmatransin PEF1-ALG2.
Although this finding is a new addition to the general artignation protein exchange mechanism, there are about 120 CUL3-binding artigins on the BTB domain, which of these artigmatins require co-aligning proteins for substrate binding, and whether the procedural mechanism mentioned by the authors is applicable to other CRL families is unknown. These remaining issues need to be further clarified.
Original link:
https://doi.org/10.1016/j.molcel.2022.01.004
Pattern Maker: Eleven
bibliography
1. Huttlin, E.L., et al. (2017). Architecture of the human interactome defines protein communities and disease networks. Nature 545, 505–509.
2. McGourty, C.A., et al. (2016). Regulation of the CUL3 Ubiquitin Ligase by a Calcium-Dependent Co-adaptor. Cell 167, 525–538.e4.
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