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Elucidation of E3 Ubiquitin Ligase Specificity Through Proteome-wide Internal Degron Mapping

ABSTRACT: The ubiquitin-proteasome system plays critical roles in biology by regulating protein degradation. Despite their importance, precise recognition specificity is known for few of the 600 E3s. Here we establish a two-pronged strategy for identifying and mapping critical residues of internal degrons on a genome scale in HEK-293T cells. We employ Global Protein Stability profiling combined with machine learning to identify 15,800 peptides likely to contain sequence-dependent degrons. We combine this with scanning mutagenesis to define critical residues for over 5,000 predicted degrons. Focusing on Cullin-RING ligase degrons, we generated mutational fingerprints for 219 degrons and developed DegronID, a computational algorithm enabling the clustering of degron peptides with similar motifs. CRISPR analysis enabled the discovery of E3-degron pairs of which we uncover 16 pairs that revealed extensive degron variability and structural determinants. We provide the visualization of this data on the public DegronID Data Browser as a resource for future exploration.

ORGANISM(S): Homo sapiens

PROVIDER: GSE240610 | GEO | 2023/09/21

REPOSITORIES: GEO

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Project description:N-degron pathway plays an important role in the protein quality control and maintenance of cellular protein homeostasis. ZER1 and ZYG11B, the substrate receptors of the Cullin 2-RING E3 ubiquitin ligase (CRL2), recognize N-terminal (Nt) glycine degrons and participate in the Nt-myristoylation quality control through the Gly/N-degron pathway. Here we show that ZER1 and ZYG11B can also recognize small Nt-residues other than glycine. Specifically, ZER1 preferentially binds to Nt-serine, -alanine, -threonine and -cysteine over -glycine, while ZYG11B prefers Nt-glycine but also has the capacity to recognize Nt-serine, -alanine and -cysteine in vitro. Nt-serine, -alanine and -cysteine undergo Nt-acetylation by NatA, thereby shielding them from recognition by ZER1/ZYG11B in cells. We found that ZER1/ZYG11B is able to target the non-acetylation of small Nt-residue degrons for degradation in NatA-deficient cells, implicating its role in the Nt-acetylation quality control. Furthermore, we present the crystal structures of ZER1 and ZYG11B bound to various small Nt-residues and uncover the molecular mechanism of non-acetylated substrate recognition by ZER1 and ZYG11B.N-degron pathway plays an important role in the protein quality control and maintenance of cellular protein homeostasis. ZER1 and ZYG11B, the substrate receptors of the Cullin 2-RING E3 ubiquitin ligase (CRL2), recognize N-terminal (Nt) glycine degrons and participate in the Nt-myristoylation quality control through the Gly/N-degron pathway. Here we show that ZER1 and ZYG11B can also recognize small Nt-residues other than glycine. Specifically, ZER1 preferentially binds to Nt-serine, -alanine, -threonine and -cysteine over -glycine, while ZYG11B prefers Nt-glycine but also has the capacity to recognize Nt-serine, -alanine and -cysteine in vitro. Nt-serine, -alanine and -cysteine undergo Nt-acetylation by NatA, thereby shielding them from recognition by ZER1/ZYG11B in cells. We found that ZER1/ZYG11B is able to target the non-acetylation of small Nt-residue degrons for degradation in NatA-deficient cells, implicating its role in the Nt-acetylation quality control. Furthermore, we present the crystal structures of ZER1 and ZYG11B bound to various small Nt-residues and uncover the molecular mechanism of non-acetylated substrate recognition by ZER1 and ZYG11B.

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Elucidation of E3 Ubiquitin Ligase Specificity Through Proteome-wide Internal Degron Mapping

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