Dissecting specificity of the human Arg/N-degron pathway
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Abstract
The Arg/N-degron pathway was the first discovered N-degron pathway in 1986 in Varshavsky's lab.
This is an important pathway for targeting proteins for degradation based on their N-terminal
residues. In this pathway, specific residues are recognized by UBR family E3 ubiquitin ligases and
marked for proteasomal degradation. This pathway plays a significant role in regulating protein
stability and function, impacting various cellular processes and diseases, including cancer,
neurodegeneration, and cardiovascular development.
It is important to focus on the N-terminal part of proteins to expand our understanding of this
pathway. For that, I tested the stability of the library containing all variants of 20 amino acids at the
first two positions (Ubi-XZ library) by global protein stability (GPS) profiling. The eK-sfGFP-P2A-
mCherry reporter, which showed the highest precision, was chosen for this purpose. I showed that
the Ubi-XZ library is highly reproducible, as evidenced by high consistency across replicates.
Besides that, the stability of fourteen constructs containing specific dipeptides compared with the
same dipeptides in the Ubi-XZ library revealed a strong correlation. The key result of the Ubi-XZ
library is the unexpected stability of N-terminal E and Q residues, which are typically targeted for
degradation by the Arg/N-degron pathway. To explore further the reasoning behind this observation I
explored various hypotheses.
Differential expression of key enzymes in the Arg/N-degron pathway might impair its functionality.
However, transcription levels of ATE1, NTAN1, NTAQ1, UBR1, UBR2, UBR4, and UBR5, which are
part of this pathway, compared with other genes in the HEK293 cell line indicated that these genes
are expressed, suggesting that the deviation is not due to a lack of these proteins. Another
hypothesis involved substrate specificity of enzymes, particularly NTAQ1, which targets N-terminal Q
for deamidation. Experiments with human NTAN1 and NTAQ1 expressed in yeast cells lacking the
Nta1 enzyme confirmed that NTAN1 and NTAQ1 are specific to their substrates.
Two studies showed that acetylation of N-terminal residues prevents degradation by the Arg/N-
degron pathway. To investigate this in the context of my results, 12 constructs with dipeptides
beginning with N, Q, E, or D and a second residue of L, R, or D were expressed in ATE1 knock out
cell line. These dipeptides underwent acetylation, matching the stability pattern in the Ubi-XZ library
and preventing the Arg/N-degron pathway from targeting N-terminal Q and N residues. Future
research should identify which NAT enzymes acetylate N-terminal E and D, with NatH as a potential
candidate.
Proteomic Peptide Phage Display (ProP-PD) identified sequences on the N-terminus of human
proteins binding to NTAN1, NTAQ1, or ATE1. An N-terminal ProP-PD library with all human protein
isoforms was used, finding 14 binders for NTAQ1 and 7 for ATE1 isoform 1. Interestingly, binders for
NTAQ1 did not start with Q, and those for ATE1 isoform 1 did not predominantly start with E or D.
Further testing the stability of these peptides in cells lacking NTAQ1 or ATE1 could reveal new
degrons.
This study highlights acetylation as a mechanism preventing degradation by the Arg/N-degron
pathway.