C-terminal amides mark proteins for degradation via SCF/FBXO31

Abstract

During normal cellular homeostasis unfolded and mis-localized proteins are recognized and removed, preventing the build-up of toxic byproducts. When protein homeostasis is perturbed during aging, neurodegeneration or cellular stress, proteins can accumulate several forms of chemical damage through reactive metabolites. Such modifications have been proposed to trigger the selective removal of chemically marked proteins; however, discovering modifications sufficient to induce protein degradation has remained challenging. Using a semi-synthetic chemical biology approach coupled to cellular assays, we found that C-terminal amide-bearing proteins (CTAPs) are rapidly cleared from human cells. A CRISPR screen identified the SCF/FBXO31 ubiquitin ligase as a reader of C-terminal amides, which ubiquitylates CTAPs for subsequent proteasomal degradation. A conserved binding pocket enables FBXO31 to bind almost any C-terminal peptide bearing an amide while retaining exquisite selectivity over non-modified clients. This mechanism facilitates binding and turnover of endogenous CTAPs that are formed following oxidative stress. A dominant human mutation found in neurodevelopmental disorders switches CTAP recognition, such that non-amidated neosubstrates are now degraded and FBXO31 becomes markedly toxic. We propose that CTAPs may represent the vanguard of a largely unexplored class of modified amino acid degrons that could provide a general strategy for selective yet broad surveillance of chemically damaged proteins.

Orphan quality control by an SCF ubiquitin ligase directed to pervasive C-degrons

“…It is striking that ~36% of random degrons (1678 out of 4709) and ~54% of degrons in the yeast C-terminome (246 out of 458) are C-degrons targeted by SCF Das1 , arguing that the SCF/C-degron pathway has a broad specificity towards C-degrons. This is in stark contrast with other C-degron pathways founds in human cells, where different degron motifs are recognized by interchangeable substrate receptors of several cullin-RING ubiquitin ligases 22,23,52,53 . Among them, Cul2-based complexes recognize multiple related C-degron motifs ending with glycine or diglycine.…”

“…As the SCF ubiquitin ligase is among the most ancient CRLs, with functions in cell cycle control conserved from yeast to mammals 58,59 , it is possible that the SCF/C-degron pathway or some of its features, such as its sequence determinants or functions in proteostasis, are conserved across eukaryotes. Supporting this possibility, the human SCF, together with the F-box substrate receptor FBXO31, appears to function as a quality control factor by targeting for degradation proteins carrying C-terminal amide degrons formed during oxidative stress 52 . Further work is needed to explore the specificity and targeting of Cdegrons by SCF complexes across model organisms, towards a comprehensive understanding of eukaryotic C-degron pathways.…”

Engineered reactivity of a bacterial E1-like enzyme enables ATP-driven modification of protein C termini

“…We anticipate that this reactivity can easily be extended to other nucleophiles for synthesis of C-terminally modified proteins. For example, ammonia could be used as a nucleophile for modification of proteins by C-terminal amidation, which has recently been shown to target proteins for ubiquitin modification by SCF/FBXO31 and proteasomal degradation 54 . Other classes of nucleophiles such as alcohols could also capture the MccA-N7G- O -AMP intermediate for installation of protein C-terminal esters, which are present in prenylated proteins including Ras GTPases 55 .…”