The N-end rule pathway is a proteolytic system in which N-terminal residues of short-lived proteins are recognized by recognition components (N-recognins) as essential components of degrons, called N-degrons. Known N-recognins in eukaryotes mediate protein ubiquitylation and selective proteolysis by the 26S proteasome. Substrates of N-recognins can be generated when normally embedded destabilizing residues are exposed at the N terminus by proteolytic cleavage. N-degrons can also be generated through modifications of posttranslationally exposed pro-N-degrons of otherwise stable proteins; such modifications include oxidation, arginylation, leucylation, phenylalanylation, and acetylation. Although there are variations in components, degrons, and hierarchical structures, the proteolytic systems based on generation and recognition of N-degrons have been observed in all eukaryotes and prokaryotes examined thus far. The N-end rule pathway regulates homeostasis of various physiological processes, in part, through interaction with small molecules. Here, we review the biochemical mechanisms, structures, physiological functions, and small-molecule-mediated regulation of the N-end rule pathway.

The N-end rule defines the protein-destabilizing activity of a given amino-terminal residue and its post-translational modification. Since its discovery 25 years ago, the pathway involved in the N-end rule has been thought to target only a limited set of specific substrates of the ubiquitin-proteasome system. Recent studies have provided insights into the components, substrates, functions and structural basis of substrate recognition. The N-end rule pathway is now emerging as a major cellular proteolytic system, in which the majority of proteins are born with or acquire specific N-terminal degradation determinants through protein-specific or global post-translational modifications.

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The N-end rule pathway is a proteolytic system in which recognition components (N-recognins) recognize a set of N-terminal residues as part of degradation signals (N-degrons). Two studies in this issue report the structures of Ubr boxes, a substrate recognition domain of eukaryotic N-recognins. We discuss how eukaryotic and prokaryotic N-recognins use a similar molecular principle to recognize a different set of N-degrons.

Summary Intracellular signaling is often mediated by a family of functionally overlapping signal mediators that contain multiple sites interacting with other proteins or ligands with weak affinity (Kd > μM). Conjugation of multiple low-affinity ligands into a high-affinity multivalent molecule provides a means to control the entire protein family within a single intracellular pathway. The N-end rule pathway is a ubiquitin (Ub)-dependent proteolytic system where at least four Ub ligases, called N-recognins, have a common domain critical for binding to type-1 (basic) and type-2 (bulky hydrophobic) destabilizing N-terminal residues of substrates as degrons. The recent development of a heterodivalent inhibitor targeting type-1 and type-2 substrate binding sites of the N-recognin family provides new opportunities to manipulate this proteolytic pathway in biochemical and pathophysiological conditions. We overview the N-end rule pathway as an intracellular target for heterodivalent molecules and discuss the basis of thermodynamics and kinetics related to heterodivalent interactions.