Neurodegeneration-Associated Protein Fragments as Short-Lived Substrates of the N-End Rule Pathway

Protein aggregates are a common feature of neurodegenerative syndromes. Specific protein fragments were found 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 disease-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

Glutamine-Specific N-Terminal Amidase, a Component of the N-End Rule Pathway

Deamidation of N-terminal Gln by Nt^Q-amidase, an N-terminal amidohydrolase, is a part of the N-end rule pathway of protein degradation. We detected the activity of Nt^Q-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 Nt^Q-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 Nt^Q-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 Nt^Q-amidase are similar to those of transglutaminases

The Auto-Generated Fragment of the Usp1 Deubiquitylase Is a Physiological Substrate of the N-End Rule Pathway

Deamidation of N-terminal Gln by the Ntaq1 Nt^Q-amidase is a part of the Arg/N-end rule pathway, a ubiquitin-dependent proteolytic system. Here we identify Gln-Usp1^(Ct), the C-terminal fragment of the autocleaved Usp1 deubiquitylase, as the first physiological Arg/N-end rule substrate that is targeted for degradation through deamidation of N-terminal Gln. Usp1 regulates genomic stability, in part through the deubiquitylation of monoubiquitylated PCNA, a DNA polymerase processivity factor. The autocleaved Usp1 remains a deubiquitylase because its fragments remain associated with Uaf1, an enhancer of Usp1 activity, until the Gln-Usp1^(Ct) fragment is selectively destroyed by the Arg/N-end rule pathway. We also show that metabolic stabilization of Gln-Usp1^(Ct) results in a decreased monoubiquitylation of PCNA and in a hypersensitivity of cells to ultraviolet irradiation. Thus, in addition to its other functions in DNA repair and chromosome segregation, the Arg/N-end rule pathway regulates genomic stability through the degradation-mediated control of the autocleaved Usp1 deubiquitylase

The N-end rule pathway counteracts cell death by destroying proapoptotic protein fragments

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

Liat1, an arginyltransferase-binding protein whose evolution among primates involved changes in the numbers of its 10-residue repeats

The arginyltransferase Ate1 is a component of the N-end rule pathway, which recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. At least six isoforms of mouse Ate1 are produced through alternative splicing of Ate1 pre-mRNA. We identified a previously uncharacterized mouse protein, termed Liat1 (ligand of Ate1), that interacts with Ate1 but does not appear to be its arginylation substrate. Liat1 has a higher affinity for the isoforms Ate1^(1A7A) and Ate1^(1B7A). Liat1 stimulated the in vitro N-terminal arginylation of a model substrate by Ate1. All examined vertebrate and some invertebrate genomes encode proteins sequelogous (similar in sequence) to mouse Liat1. Sequelogs of Liat1 share a highly conserved ∼30-residue region that is shown here to be required for the binding of Liat1 to Ate1. We also identified non-Ate1 proteins that interact with Liat1. In contrast to Liat1 genes of nonprimate mammals, Liat1 genes of primates are subtelomeric, a location that tends to confer evolutionary instability on a gene. Remarkably, Liat1 proteins of some primates, from macaques to humans, contain tandem repeats of a 10-residue sequence, whereas Liat1 proteins of other mammals contain a single copy of this motif. Quantities of these repeats are, in general, different in Liat1 of different primates. For example, there are 1, 4, 13, 13, 17, and 17 repeats in the gibbon, gorilla, orangutan, bonobo, neanderthal, and human Liat1, respectively, suggesting that repeat number changes in this previously uncharacterized protein may contribute to evolution of primates

Downregulation of the Arg/N-degron Pathway Sensitizes Cancer Cells to Chemotherapy In Vivo

© 2020 The American Society of Gene and Cell Therapy With the use of a small interfering RNA (siRNA)-mediated approach for selective downregulation of the Arg/N-degron pathway in a murine hepatocellular carcinoma model, Leboeuf et al. demonstrated potentiated action of chemotherapy. Since components of this pathway are ubiquitously expressed, targeting the N-degron pathway has the potential to increase effectiveness of therapy in many cancer types