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In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
PAM forms an atypical SCF ubiquitin ligase complex that ubiquitinates and degrades NMNAT2
Muriel Desbois1, Oliver Crawley1, Paul R Evans2
1From the Department of Neuroscience, The Scripps Research Institute, Scripps Florida, Jupiter, Florida 33458 and.
Insights
PHR proteins like PAM are E3 ubiquitin ligases crucial for nerve development and degeneration. This study reveals how PAM forms a unique SCF complex with FBXO45 and SKP1 to polyubiquitinate and degrade NMNAT2, impacting axon stability.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- PHR (PAM/Highwire/RPM-1) proteins are conserved RING E3 ubiquitin ligases involved in neuronal development and degeneration.
- NMNAT2 is a key mediator in PHR protein-driven axon degeneration, but the inhibitory mechanism remains unclear.
Purpose of the Study:
- To elucidate the biochemical mechanism by which the PHR protein PAM (MYCBP2) interacts with and regulates NMNAT2.
- To characterize the composition and function of the noncanonical SCF complex formed by PAM.
Main Methods:
- Biochemical assays to determine complex formation and protein-protein interactions.
- Ubiquitination assays to assess NMNAT2 modification by PAM.
- Proteasome-mediated degradation studies.
Main Results:
- PAM forms a noncanonical SCF complex with FBXO45 and SKP1, lacking CUL1.
- FBXO45 is essential for the assembly of the PAM/FBXO45/SKP1 complex.
- SKP1 acts as an auxiliary component, enhancing FBXO45 binding to NMNAT2.
- PAM directly polyubiquitinates NMNAT2, leading to its proteasomal degradation and regulation of protein stability.
Conclusions:
- The study deciphers the molecular mechanism of NMNAT2 regulation by the PHR protein PAM.
- PAM utilizes a noncanonical SCF complex to target NMNAT2 for ubiquitination and degradation, impacting axon degeneration pathways.
Abstract:
PHR (PAM/Highwire/RPM-1) proteins are conserved RING E3 ubiquitin ligases that function in developmental processes, such as axon termination and synapse formation, as well as axon degeneration. At present, our understanding of how PHR proteins form ubiquitin ligase complexes remains incomplete. Although genetic studies indicate NMNAT2 is an important mediator of PHR protein function in axon degeneration, it remains unknown how PHR proteins inhibit NMNAT2. Here, we decipher the biochemical basis for how the human PHR protein PAM, also called MYCBP2, forms a noncanonical Skp/Cullin/F-box (SCF) complex that contains the F-box protein FBXO45 and SKP1 but lacks CUL1. We show FBXO45 does not simply function in substrate recognition but is important for assembly of the PAM/FBXO45/SKP1 complex. Interestingly, we demonstrate a novel role for SKP1 as an auxiliary component of the target recognition module that enhances binding of FBXO45 to NMNAT2. Finally, we provide biochemical evidence that PAM polyubiquitinates NMNAT2 and regulates NMNAT2 protein stability and degradation by the proteasome.
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