Review coordinated by Life Science Editors Foundation

Reviewed by: Dr. Angela Andersen, Life Science Editors Foundation & Life Science Editors. *Assisted substantially by NotebookLM.

Potential Conflicts of Interest: Angela thinks Olivia Rissland is everything a scientist should be.

What is an N-degron? N-degrons are short amino acid sequences located at the N-terminus of a protein that signal for the protein's degradation. This process is an essential part of protein quality control and regulation within cells. N-degrons are recognized by specific E3 ubiquitin ligases, also known as N-recognins, which help target the protein for degradation by the ubiquitin-proteasome system.

How was this new Arg/N-degron pathway discovered? The authors were initially studying how N-terminal sequences affect gene expression using a reporter gene assay. They found that a specific tripeptide motif (KIH) inserted at the N-terminus of a reporter protein led to a dramatic decrease in protein expression. Further investigation revealed that this decrease was due to rapid protein degradation, indicating the presence of a novel N-degron.

What are the key features of this new N-degron pathway? This newly discovered N-degron pathway targets proteins with a lysine (K) or arginine (R) residue at the third position (position 3) from the N-terminus. Importantly, this pathway requires: * • Methionine Removal: The initiator methionine (M) at position 1 must be removed by the enzyme methionine aminopeptidase 2 (MetAP2) for the degron to be active. * • UBR4 Recognition: The E3 ligase UBR4, but not UBR1 or UBR2, recognizes this specific degron and initiates the degradation process.

Why is the identity of the second amino acid important? The second amino acid plays a crucial role in determining whether MetAP2 can cleave the initiator methionine. This study found that the degron is only active when the second amino acid is threonine (T) or valine (V). These amino acids allow MetAP2 to remove the methionine, exposing the lysine or arginine at position 3 for recognition by UBR4. In contrast, if the second amino acid is alanine (A) or serine (S), MetAP1 removes the methionine. The researchers hypothesize that these N-termini are then acetylated, preventing UBR4 recognition.

Is there evidence that this pathway affects endogenous proteins? Yes, analysis of previously published data and additional experiments by the researchers suggest that this MetAP2-UBR4 pathway is not limited to artificial reporter systems. They found that endogenous proteins with MTK or MVK N-termini were less stable than those with other amino acids at position.

Does UBR4 work alone in this pathway? UBR4 appears to function as part of a complex with the protein KCMF1 to degrade proteins containing this new degron. Experiments showed that disrupting the UBR4-KCMF1 complex stabilized the degradation of reporter proteins containing the KIH degron.

What is the broader significance of this discovery? The identification of this new Arg/N-degron pathway expands our understanding of the N-end rule, a fundamental mechanism for protein degradation in cells. It highlights the complexity of this system and reveals how the interplay between different enzymes like MetAP2 and E3 ligases like UBR4 can fine-tune protein stability. Additionally, it suggests that there may be other undiscovered N-degron pathways that remain to be characterized.

What questions still need to be answered about this new pathway? This study raises several new questions, including: * • Substrate Specificity: What are the precise rules governing UBR4 recognition of position 3 lysine and arginine degrons? Do other amino acids in the protein sequence affect degron recognition? * • Physiological Roles: What are the specific cellular processes and pathways regulated by this MetAP2-UBR4 N-degron pathway? * • Evolutionary Conservation: Is this pathway conserved in other organisms, or is it unique to mammals? * • Therapeutic Potential: Could this pathway be targeted for therapeutic purposes? For example, could stabilizing proteins involved in disease by manipulating this pathway be beneficial?

What was not known: * • Whether a lysine or arginine residue at position 3 of a protein could act as an N-degron. * • Whether MetAP2 could play a role in initiating N-degron-mediated degradation.

What this preprint reveals: * • A new family of N-degrons: The study identified a new class of N-degrons characterized by a lysine or arginine residue at position 3, following a methionine at position 1 and a MetAP2-cleavable residue (threonine or valine) at position * • MetAP2-dependent initiation of the Arg/N-degron pathway: The study found that MetAP2-mediated removal of the initiator methionine is essential for the recognition and degradation of these position 3 lysine/arginine degrons. This is the first demonstration of MetAP2's involvement in this pathway * • UBR4 as the primary E3 ligase: UBR4, rather than UBR1 or UBR2, was identified as the primary E3 ligase responsible for recognizing and targeting proteins with the newly identified position 3 degrons for degradation. * • Role of downstream residues: The study showed that amino acid residues downstream of the position 3 lysine/arginine can influence both methionine cleavage by MetAP2 and recognition by UBR4, highlighting the complexity of the N-degron pathway. * • Endogenous protein regulation: The study provided evidence suggesting that this MetAP2-dependent, UBR4-mediated Arg/N-degron pathway regulates the stability of endogenous proteins, highlighting its broader biological significance.

Ang's take- somewhat specialized and 'ectopic' but important, thorough, and unambiguous. Satisfying. Very likely to be physiologically relevant even though most of the assays were done with reporters. Regardless, showing that this rule 'is' true is useful for technological applications.