In the course of apoptosis, activated caspases cleave ∼500 to ∼1,000 different proteins in a mammalian cell. The dynamics of apoptosis involve a number of previously identified, caspase-generated proapoptotic protein fragments, defined as those that increase the probability of apoptosis. In contrast to activated caspases, which can be counteracted by inhibitor of apoptosis proteins, there is little understanding of antiapoptotic responses to proapoptotic protein fragments. One possibility is the regulation of proapoptotic fragments through their selective degradation. The previously identified proapoptotic fragments Cys-RIPK1, Cys-TRAF1, Asp-BRCA1, Leu-LIMK1, Tyr-NEDD9, Arg-BID, Asp-BCL XL , Arg-BIM EL , Asp-EPHA4, and Tyr-MET bear destabilizing N-terminal residues. Tellingly, the destabilizing nature (but not necessarily the actual identity) of N-terminal residues of proapoptotic fragments was invariably conserved in evolution. Here, we show that these proapoptotic fragments are short-lived substrates of the Arg/N-end rule pathway. Metabolic stabilization of at least one such fragment, Cys-RIPK1, greatly augmented the activation of the apoptosis-inducing effector caspase-3. In agreement with this understanding, even a partial ablation of the Arg/N-end rule pathway in two specific N-end rule mutants is shown to sensitize cells to apoptosis. We also found that caspases can inactivate components of the Arg/N-end rule pathway, suggesting a mutual suppression between this pathway and proapoptotic signaling. Together, these results identify a mechanistically specific and functionally broad antiapoptotic role of the Arg/N-end rule pathway. In conjunction with other apoptosis-suppressing circuits, the Arg/N-end rule pathway contributes to thresholds that prevent a transient or otherwise weak proapoptotic signal from reaching the point of commitment to apoptosis.

Substrates of a ubiquitin-dependent proteolytic system called the N-end rule pathway include proteins with destabilizing N-terminal residues. N-recognins, the pathway's ubiquitin ligases, contain three substrate-binding sites. The type-1 site is specific for basic N-terminal residues (Arg, Lys, and His). The type-2 site is specific for bulky hydrophobic N-terminal residues (Trp, Phe, Tyr, Leu, and Ile). We show here that the type-1/2 sites of UBR1, the sole N-recognin of the yeast Saccharomyces cerevisiae, are located in the first ϳ700 residues of the 1,950-residue UBR1. These sites are distinct in that they can be selectively inactivated by mutations, identified through a genetic screen. Mutations inactivating the type-1 site are in the previously delineated ϳ70-residue UBR motif characteristic of N-recognins. Fluorescence polarization and surface plasmon resonance were used to determine that UBR1 binds, with a K d of ϳ1 M, to either type-1 or type-2 destabilizing N-terminal residues of reporter peptides but does not bind to a stabilizing N-terminal residue such as Gly. A third substrate-binding site of UBR1 targets an internal degron of CUP9, a transcriptional repressor of peptide import. We show that the previously demonstrated in vivo dependence of CUP9 ubiquitylation on the binding of cognate dipeptides to the type-1/2 sites of UBR1 can be reconstituted in a completely defined in vitro system. We also found that purified UBR1 and CUP9 interact nonspecifically and that specific binding (which involves, in particular, the binding by cognate dipeptides to the UBR1 type-1/2 sites) can be restored either by a chaperone such as EF1A or through macromolecular crowding.

SUMMARY Protein aggregates are a common feature of neurodegenerative syndromes. Specific protein fragments were found previously to be aggregated in disorders including Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease. Here we show that the natural C-terminal fragments of Tau, TDP43, and α-synuclein are short-lived substrates of the Arg/N-end rule pathway, a processive proteolytic system that targets proteins bearing “destabilizing” N-terminal residues. Furthermore, a natural TDP43 fragment is shown to be metabolically stabilized in Ate1−/− fibroblasts that lack the arginylation branch of the Arg/N-end rule pathway, leading to accumulation and aggregation of this fragment. We also found that a fraction of Aβ42, the Alzheimer’s-associated fragment of APP, is N-terminally arginylated in the brains of 5xFAD mice and is degraded by the Arg/N-end rule pathway. The discovery that neurodegeneration-associated natural fragments of TDP43, Tau, α-synuclein, and APP can be selectively destroyed by the Arg/N-end rule pathway suggests that this pathway counteracts neurodegeneration.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Primary destabilizing N-terminal residues (Nd p ) are recognized directly by the targeting machinery. The recognition of secondary destabilizing N-terminal residues (Nd s ) is preceded by conjugation of an Nd p residue to Nd s of a polypeptide substrate. In eukaryotes, ATE1 -encoded arginyl-transferases (R D,E,C* -transferases) conjugate Arg (R), an Nd p residue, to Nd s residues Asp (D), Glu (E), or oxidized Cys residue (C*). Ubiquitin ligases recognize the N-terminal Arg of a substrate and target the (ubiquitylated) substrate to the proteasome. In prokaryotes such as Escherichia coli , Nd p residues Leu (L) or Phe (F) are conjugated, by the aat -encoded Leu/Phe-transferase (L/F K,R -transferase), to N-terminal Arg or Lys, which are Nd s in prokaryotes but Nd p in eukaryotes. In prokaryotes, substrates bearing the Nd p residues Leu, Phe, Trp, or Tyr are degraded by the proteasome-like ClpAP protease. Despite enzymological similarities between eukaryotic R D,E,C* -transferases and prokaryotic L/F K,R -transferases, there is no significant sequelogy (sequence similarity) between them. We identified an aminoacyl-transferase, termed Bpt, in the human pathogen Vibrio vulnificus . Although it is a sequelog of eukaryotic R D,E,C* -transferases, this prokaryotic transferase exhibits a “hybrid” specificity, conjugating Nd p Leu to Nd s Asp or Glu. Another aminoacyl-transferase, termed ATEL1, of the eukaryotic pathogen Plasmodium falciparum , is a sequelog of prokaryotic L/F K,R -transferases (Aat), but has the specificity of eukaryotic R D,E,C* -transferases (ATE1). Phylogenetic analysis suggests that the substrate specificity of R-transferases arose by two distinct routes during the evolution of eukaryotes.

SUMMARY Deamidation of N-terminal Gln by NtQ-amidase, a previously undescribed N-terminal amidohydrolase, is a part of the N-end rule pathway of protein degradation. We detected the activity of NtQ-amidase, termed Ntaq1, in mouse tissues, purified Ntaq1 from bovine brains, identified its gene, and began analyzing this enzyme. Ntaq1 is highly conserved among animals, plants and some fungi, but its sequence is dissimilar to sequences of other amidases. An earlier mutant in the Drosophila Cg8253 gene that we show here to encode NtQ-amidase has defective long-term memory. Other studies identified protein ligands of the uncharacterized human C8orf32 protein that we show here to be the Ntaq1 NtQ-amidase. Remarkably, “high-throughput” studies have recently solved the crystal structure of C8orf32 (Ntaq1). Our site-directed mutagenesis of Ntaq1 and its crystal structure indicate that the active site and catalytic mechanism of NtQ-amidase are similar to those of transglutaminases.