Rule Pathway Research Articles

Arginyl-tRNA protein transferase 1 (ATE1) regulates protein degradation by Arg/N-end rule pathway

Previous studies have shown that deletion or downregulation of ATE1 can lead to neurodegeneration, behavioral abnormalities, hyperphagia and lower body weight. To determine how loss of ATE1 affects energy metabolism and food intake, ATE1-deficient and ATE1-containing mice were given high fat diet for 12 weeks. Based on our current data, we found significant sex differences in body weight and food intakes, with ATE1-deficient mice less sensitive to the obesogenic effects of high fat diet even while consuming the same amount of high fat diet as controls. Interestingly, ATE1-deficient mice exposed to high fat diet displayed lower plasma leptin levels. Leptin-treated ATE1-deficient mice show changes in pSTAT3 expression in the arcuate nucleus of the hypothalamus relative to leptin-treated ATE1-containing mice. Lastly, diet-induced obesity can be ameliorated by tamoxifen-induced knockout of ATE1 in adult male mice relative to vehicle-treated WT mice. Taken together, these data indicate that loss of ATE1 produces attenuated leptin levels in mouse models of diet-induced obesity and can mitigate the effects of high fat diet. NIGMS, 1SC1GM144190-01 (ESN), NINDS R15NS095317 (CSB), Texas Woman's University internal funds. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Insights into the recognition mechanism in the UBR box of UBR4 for its specific substrates

The N-end rule pathway is a proteolytic system involving the destabilization of N-terminal amino acids, known as N-degrons, which are recognized by N-recognins. Dysregulation of the N-end rule pathway results in the accumulation of undesired proteins, causing various diseases. The E3 ligases of the UBR subfamily recognize and degrade N-degrons through the ubiquitin-proteasome system. Herein, we investigated UBR4, which has a distinct mechanism for recognizing type-2 N-degrons. Structural analysis revealed that the UBR box of UBR4 differs from other UBR boxes in the N-degron binding sites. It recognizes type-2 N-terminal amino acids containing an aromatic ring and type-1 N-terminal arginine through two phenylalanines on its hydrophobic surface. We also characterized the binding mechanism for the second ligand residue. This is the report on the structural basis underlying the recognition of type-2 N-degrons by the UBR box with implications for understanding the N-end rule pathway.

Pharmacologic targeting of the p62 ZZ domain enhances both anti-tumor and bone-anabolic effects of bortezomib in multiple myeloma.

Multiple myeloma (MM) is a malignancy of plasma cells whose antibody secretion creates proteotoxic stress relieved by the N-end rule pathway, a proteolytic system that degrades N-arginylated proteins in the proteasome. When the proteasome is inhibited, protein cargo is alternatively targeted for autophagic degradation by binding to the ZZ-domain of p62/ sequestosome-1. Here, we demonstrate that XRK3F2, a selective ligand for the ZZ-domain, dramatically improved two major responses to the proteasome inhibitor bortezomib (Btz) by increasing: i) killing of human MM cells by stimulating both Btz-mediated apoptosis and necroptosis, a process regulated by p62; and ii) preservation of bone mass by stimulating osteoblast differentiation and inhibiting osteoclastic bone destruction. Co-administration of Btz and XRK3F2 inhibited both branches of the bimodal N-end rule pathway exhibited synergistic anti-MM effects on MM cell lines and CD138+ cells from MM patients, and prevented stromal-mediated MM cell survival. In mice with established human MM, co-administration of Btz and XRK3F2 decreased tumor burden and prevented the progression of MM-induced osteolytic disease by inducing new bone formation more effectively than either single agent alone. The results suggest that p62-ZZ ligands enhance the anti- MM efficacy of proteasome inhibitors and can reduce MM morbidity and mortality by improving bone health.

Animal Model Considerations for Medical Countermeasure Development for Radiation and Sulfur Mustard Exposures: Animal models for radiation and HD exposures.

Development of medical countermeasures (MCM) to mitigate and/ or treat the pulmonary complications associated with exposure to chemical, radiological, and/ or nuclear weapons is a national, public health preparedness posture priority in the United States (US). Pulmonary exposure to either sulfur mustard vapor or radiation causes oxidative damage, vascular injury, hyperinflammation, and pro-fibrotic signaling cascades that lead to life-threatening and potentially debilitating lung disease. There is no MCM currently approved by the US Food and Drug Administration (FDA) to mitigate and/ or treat lung injury caused by sulfur mustard or radiation exposure. Thus, there remains a major unmet public health need for development of threat-agnostic, host-directed therapeutics that target common pathophysiological mechanisms underlying the progression of acute and/ or late lung injury independent of the etiology of disease. This review describes the clinical manifestations and underlying mechanisms of sulfur mustard and radiation-induced lung injury and regulatory considerations for MCM development under the non-traditional Animal Rule pathway.

The N-end rule pathway regulates ER stress-induced clusterin release to the cytosol where it directs misfolded proteins for degradation

Previous work suggests that cell stress induces release of the normally secreted chaperone clusterin (CLU) into the cytosol. We analyzed the localization of CLU in healthy and stressed cells, the mechanism of its cytosolic release, and its interactions with cytosolic misfolded proteins. Key results of this study are the following: (1) full-length CLU is released to the cytosol during stress, (2) the CLU N-terminal D1 residue is recognized by the N-end rule pathway and together with the enzyme ATE1 is essential for cytosolic release, (3) CLU can form stable complexes with cytosolic misfolded proteins and direct them to the proteasome and autophagosomes, and (4) cytosolic CLU protects cells from hypoxic stress and the cytosolic overexpression of an aggregation-prone protein. Collectively, the results suggest that enhanced cytosolic release of CLU is a stress response that can inhibit the toxicity of misfolded proteins and facilitate their targeted degradation via both autophagy and the proteasome.

Single amino acid–based PROTACs trigger degradation of the oncogenic kinase BCR–ABL in chronic myeloid leukemia (CML)

Proteolysis-targeting chimera (PROTAC) that specifically targets harmful proteins for destruction by hijacking the ubiquitin-proteasome system is emerging as a potent anticancer strategy. How to efficiently modulate the target degradation remains a challenging issue. In this study, we employ a single amino acid-based PROTAC, which uses the shortest degradation signal sequence as the ligand of the N-end rule E3 ubiquitin ligases to degrade the fusion protein BCR (breakpoint cluster region)-ABL (Abelson proto-oncogene), an oncogenic kinase that drives the progression of chronic myeloid leukemia. We find that the reduction level of BCR-ABL can be easily adjusted by substituting different amino acids. Furthermore, a single PEG linker is found to achieve the best proteolytic effect. Our efforts have resulted in effective degradation of BCR-ABL protein by the N-end rule pathway and efficient growth inhibition of K562cells expressing BCR-ABL invitro and blunted tumor growth in a K562 xenograft tumor model invivo. The PROTAC presented has unique advantages including lower effective concentration, smaller molecular size, and modular degradation rate. Demonstrating the efficacy of the N-end rule-based PROTACs invitro and invivo, our study further expands the limited degradation pathways currently available for PROTACs invivo and is easily adapted for broader applications in targeted protein degradation.

Assays for the Evaluation of the Immune Response to Marburg and Ebola Sudan Vaccination-Filovirus Animal Nonclinical Group Anti-Marburg Virus Glycoprotein Immunoglobulin G Enzyme-Linked Immunosorbent Assay and a Pseudovirion Neutralization Assay.

Since the discovery of the Marburg virus (MARV) in 1967 and Ebola virus (EBOV) in 1976, there have been over 40 reported outbreaks of filovirus disease with case fatality rates greater than 50%. This underscores the need for efficacious vaccines against these highly pathogenic filoviruses. Due to the sporadic and unpredictable nature of filovirus outbreaks, such a vaccine would likely need to be vetted through the U.S. Food and Drug Administration (FDA), following the Animal Rule or similar European Medicines Agency (EMA) regulatory pathway. Under the FDA Animal Rule, vaccine-induced immune responses correlating with survival of non-human primates (NHPs), or another well-characterized animal model, following lethal challenge, will need to be bridged for human immune response distributions in clinical trials. A correlate of protection has not yet been identified for the filovirus disease, but antibodies, specifically anti-glycoprotein (GP) antibodies, are believed to be critical in providing protection against the filovirus disease following vaccination and are thus a strong candidate for a correlate of protection. Thus, species-neutral methods capable of the detection and bridging of these antibody immune responses, such as methods to quantify anti-GP immunoglobulin G (IgG)-binding antibodies and neutralizing antibodies, are needed. Reported here is the development and qualification of two Filovirus Animal Nonclinical Group (FANG) anti-GP IgG Enzyme-Linked Immunosorbent Assays (ELISAs) to quantify anti-MARV and anti-Sudan virus (SUDV) IgG antibodies in human and NHP serum samples, as well as the development of pseudovirion neutralization assays (PsVNAs) to quantify MARV- and SUDV-neutralizing antibodies in human and NHP serum samples.

The development of broad-spectrum antiviral medical countermeasures to treat viral hemorrhagic fevers caused by natural or weaponized virus infections.

The Joint Program Executive Office for Chemical, Biological, Radiological, and Nuclear Defense (JPEO-CBRND) began development of a broad-spectrum antiviral countermeasure against deliberate use of high-consequence viral hemorrhagic fevers (VHFs) in 2016. The effort featured comprehensive preclinical research, including laboratory testing and rapid advancement of lead molecules into nonhuman primate (NHP) models of Ebola virus disease (EVD). Remdesivir (GS-5734, Veklury, Gilead Sciences) was the first small molecule therapeutic to successfully emerge from this effort. Remdesivir is an inhibitor of RNA-dependent RNA polymerase, a viral enzyme that is essential for viral replication. Its robust potency and broad-spectrum antiviral activity against certain RNA viruses including Ebola virus and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) led to its clinical evaluation in randomized, controlled trials (RCTs) in human patients during the 2018 EVD outbreak in the Democratic Republic of the Congo (DRC) and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic today. Remdesivir was recently approved by the US Food and Drug Administration (FDA) for the treatment of COVID-19 requiring hospitalization. Substantial gaps remain in improving the outcomes of acute viral infections for patients afflicted with both EVD and COVID-19, including how to increase therapeutic breadth and strategies for the prevention and treatment of severe disease. Combination therapy that joins therapeutics with complimentary mechanisms of action appear promising, both preclinically and in RCTs. Importantly, significant programmatic challenges endure pertaining to a clear drug and biological product development pathway for therapeutics targeting biodefense and emerging pathogens when human efficacy studies are not ethical or feasible. For example, remdesivir’s clinical development was facilitated by outbreaks of Ebola and SARS-CoV-2; as such, the development pathway employed for remdesivir is likely to be the exception rather than the rule.The current regulatory licensure pathway for therapeutics targeting rare, weaponizable VHF agents is likely to require use of FDA’s established Animal Rule (21 CFR 314.600–650 for drugs; 21 CFR 601.90–95 for biologics). The FDA may grant marketing approval based on adequate and well-controlled animal efficacy studies when the results of those studies establish that the drug is safe and likely to produce clinical benefit in humans. In practical terms, this is anticipated to include a series of rigorous, well-documented, animal challenge studies, to include aerosol challenge, combined with human safety data. While small clinical studies against naturally occurring, high-consequence pathogens are typically performed where possible, approval for the therapeutics currently under development against biodefense pathogens will likely require the Animal Rule pathway utilizing studies in NHPs. We review the development of remdesivir as illustrative of the effort that will be needed to field future therapeutics against highly lethal, infectious agents.

Structural insight into N-degron polyubiquitination catalyzed by Ubr1

The N-end rule pathway was one of the first ubiquitin (Ub)-dependent degradation pathways to be identified. Ubr1, a single-chain E3 ligase, targets proteins bearing a destabilizing residue at the N-terminus (N-degron) for rapid K48-linked ubiquitination and proteasome-dependent degradation. How Ubr1 catalyses the initiation of ubiquitination on the substrate and elongation of the Ub chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here, we report the cryo-electron microscopy structures of two complexes representing the initiation and elongation intermediates of Ubr1 captured using chemical approaches.

Cereblon covalent modulation through structure-based design of histidine targeting chemical probes.

Electrophilic biocompatible warheads, particularly cysteine-reactive acrylamides, have enabled the development of covalent inhibitor drugs and chemical biology probes, but cysteine is rarely present in protein binding sites. Therefore, expansion of the list of targetable amino acid residues is required to augment the synthetic bology toolkit of site-selective protein modifications. This work describes the first rational targeting of a specific histidine residue in a protein binding site using sulfonyl exchange chemistry. Structure-based drug design was used to incorporate sulfonyl fluoride and triazole reactive groups into the isoindolinone thalidomide congener EM12 to yield potent covalent inhibitors of the cereblon E3 ubiquitin ligase complex through engagement of His353. Conversely, the fluorosulfate derivative EM12-FS labels His353, but degrades a novel neosubstrate, the protein N-terminal glutamine amidohydrolase NTAQ1, which is involved in the N-end rule pathway and DNA damage response. Targeted protein degradation using cereblon ligands has become an important new drug discovery modality and the chemical probes and covalent labeling strategy described here will broadly impact this exciting area of therapeutic research.

Physiological Role of Aerobic Fermentation Constitutively Expressed in an Aluminum-Tolerant Cell Line of Tobacco (Nicotiana tabacum)

Aluminum (Al)-tolerant tobacco cell line ALT301 derived from SL (wild-type) hardly exhibits Al-triggered reactive oxygen species (ROS) compared with SL. Molecular mechanism leading to this phenotype was investigated comparatively with SL. Under normal growth condition, metabolome data suggested the activation of glycolysis and lactate fermentation but the repression of the tricarboxylic acid (TCA) cycle in ALT301, namely aerobic fermentation, which seemed to be transcriptionally controlled partly by higher expression of genes encoding lactate dehydrogenase and pyruvate dehydrogenase kinase. Microarray and gene ontology analyses revealed the upregulation of the gene encoding related to APETALA2.3 (RAP2.3)-like protein, one of the group VII ethylene response factors (ERFVIIs), in ALT301. ERFVII transcription factors are known to be key regulators for hypoxia response that promotes substrate-level ATP production by glycolysis and fermentation. ERFVIIs are degraded under normoxia by the N-end rule pathway of proteolysis depending on both oxygen and nitric oxide (NO), and NO is produced mainly by nitrate reductase (NR) in plants. In ALT301, levels of the NR gene expression (NIA2), NR activity and NO production were all lower compared with SL. Consistently, the known effects of NO on respiratory pathways were also repressed in ALT301. Under Al-treatment condition, NO level increased in both lines but was lower in ALT301. These results suggest that the upregulation of the RAP2.3-like gene and the downregulation of the NIA2 gene and resultant NO depletion in ALT301 coordinately enhance aerobic fermentation, which seems to be related to a higher capacity to prevent ROS production in mitochondria under Al stress.

Recent advances in protein modifications techniques for the targeting N‐terminal cysteine

AbstractN‐terminal modifications of proteins have garnered the attention of chemical biologists because of their critical roles in numerous cellular processes, including protein translocation, protein structural and metabolic stability, and the regulation of the half‐life of proteins by a process known as “N‐end rule pathway.” In addition, other posttranslational modification processes also depend on the N‐terminal signal sequences. Chemical probes are powerful tools that can be used to investigate N‐terminal modifications, to gain structural and functional insights into protein modification, protein‐protein interactions, protein localization, and protein dynamics. Most modifications target cysteine and lysine residues because of their high nucleophilicity. However, due to multiple occurrences of these residues in a protein at a given time, regioselective labeling can be quite difficult to accomplish. N‐terminal cysteine presents itself as a unique practical option to overcome regioselectivity and site‐specificity challenges. This review provides an overview of N‐terminal cysteine labeling tools, including N‐terminal cysteine condensation with aldehydes, cyanobenzothiazoles, cyclopropenones, and trans‐thioesterification. The review also highlights the technical challenges of each of the techniques, which need to be addressed to broaden the scope of these approaches.

Arginyltransferase1 mediates cell death by a mitochondria‐dependent pathway

The regulation of cell death in eukaryotic cells is a fundamental process for the adaptation to stress or for the response to signals. Recent studies showed that an evolutionarily conserved enzyme, arginyltransferase1 (ATE1), which is known to induce protein degradation via the N-end rule pathway, is also involved in cell death. Particularly, ATE1 is elevated under acute oxidative stress conditions and is causing apoptosis in yeast and mammalian cells. However, how does ATE1 mediate cell death remains unknown. Here, by using budding yeast as a test model, we found that ATE1 is colocalized with mitochondria under oxidative stress, and that this translocation is essential for ATE1-induced apoptosis. We also found that the mutations which disrupt the arginylation activity also compromise the ATE1-induced cell death, without affecting its mitochondrial localization. Furthermore, we found that the ATE1-mediated cell death is not dependent on mitochondrial ETC activity or the caspase pathway. Rather, we found that ATE1-mediated cell death is dependent on the mitochondrial permeability pore and the apoptosis-inducing factor. We conclude that ATE1 is a novel apoptosis-regulator that utilizes specific mitochondrial pathways.

The E3-ligase AoUBR1 in N-end rule pathway is involved in the vegetative growth, secretome, and trap formation in Arthrobotrys oligospora

The N-end rule pathway is a regulated protein degradation system. Arthrobotrys oligospora, a typical nematode-trapping fungus, switches its life strategies from saprophytism to carnivorism when capturing free-living nematodes by means of adhesive networks. In this study, a putative E3-ligase AoUBR1 involved in N-end rule pathway was characterized in A. oligospora during vegetative growth and trap formation. Expression of AoUBR1 coding gene was down-regulated during trap formation. Compared with wild type, the AoUBR1 knock-out mutants decreased the vegetative growth, formed less traps, and turned to be sensitive to cold stress, while, AoUBR1 overexpression mutants lost the capacity to produce conidia and also formed less traps. A number of genes differentially expressed by knock-out and overexpression of AoUBR1, which lead to the transcriptional responses associated with plasma membrane, transportation, oxidation, and proteolysis. AoUBR1 knock-out also resulted in the down-regulation of numerous secreted proteins associated with carnivorism and nutrient utilization from nematodes. In addition, AoUBR1 homologs were conserved in nematode-trapping fungi based on the genome searching. Therefore, the results suggested AoUBR1 in A. oligospora and its homologs in other trapping fungi are involved in the lifestyle switch between saprophytism and carnivorism.

Exploring the Complexity of Protein-Level Dosage Compensation that Fine-Tunes Stoichiometry of Multiprotein Complexes.

Proper control of gene expression levels upon various perturbations is a fundamental aspect of cellular robustness. Protein-level dosage compensation is one mechanism buffering perturbations to stoichiometry of multiprotein complexes through accelerated proteolysis of unassembled subunits. Although N-terminal acetylation- and ubiquitin-mediated proteasomal degradation by the Ac/N-end rule pathway enables selective compensation of excess subunits, it is unclear how widespread this pathway contributes to stoichiometry control. Here we report that dosage compensation depends only partially on the Ac/N-end rule pathway. Our analysis of genetic interactions between 18 subunits and 12 quality control factors in budding yeast demonstrated that multiple E3 ubiquitin ligases and N-acetyltransferases are involved in dosage compensation. We find that N-acetyltransferases-mediated compensation is not simply predictable from N-terminal sequence despite their sequence specificity for N-acetylation. We also find that the compensation of Pop3 and Bet4 is due in large part to a minor N-acetyltransferase NatD. Furthermore, canonical NatD substrates histone H2A/H4 were compensated even in its absence, suggesting N-acetylation-independent stoichiometry control. Our study reveals the complexity and robustness of the stoichiometry control system.

Overexpression of Jatropha curcas ERFVII2 Transcription Factor Confers Low Oxygen Tolerance in Transgenic Arabidopsis by Modulating Expression of Metabolic Enzymes and Multiple Stress-Responsive Genes.

Enhancing crop tolerance to waterlogging is critical for improving food and biofuel security. In waterlogged soils, roots are exposed to a low oxygen environment. The group VII ethylene response factors (ERFVIIs) were recently identified as key regulators of plant low oxygen response. Oxygen-dependent N-end rule pathways can regulate the stability of ERFVIIs. This study aims to characterize the function of the Jatropha curcas ERFVIIs and the impact of N-terminal modification that stabilized the protein toward low oxygen response. This study revealed that all three JcERFVII proteins are substrates of the N-end rule pathway. Overexpression of JcERFVII2 conferred tolerance to low oxygen stress in Arabidopsis. In contrast, the constitutive overexpression of stabilized JcERFVII2 reduced low oxygen tolerance. RNA-seq was performed to elucidate the functional roles of JcERFVII2 and the impact of its N-terminal modification. Overexpression of both wildtype and stabilized JcERFVII2 constitutively upregulated the plant core hypoxia-responsive genes. Besides, overexpression of the stabilized JcERFVII2 further upregulated various genes controlling fermentative metabolic processes, oxidative stress, and pathogen responses under aerobic conditions. In summary, JcERFVII2 is an N-end rule regulated waterlogging-responsive transcription factor that modulates the expression of multiple stress-responsive genes; therefore, it is a potential candidate for molecular breeding of multiple stress-tolerant crops.

Chimera induced protein degradation: PROTACs and beyond.

Ubiquitin-proteasome system, autophagy-lysosome pathway and N-end rule pathway are crucial protein quality control mechanisms in human body. Hijacking these endogenous protein degrading measures by chimera degraders could be a revolutionary strategy for the discovery of small-molecule drugs. As the most advanced chimera degraders, PROTACs have demonstrated the potential by delivering two drug candidates into clinical trials. The development of chimera degraders exploiting these three pathways are reviewed, a focus is given on the chemical structures and their influences on biological effects from a viewpoint of medicinal chemistry.

Tying up loose ends: the N-degron and C-degron pathways of protein degradation.

Selective protein degradation by the ubiquitin-proteasome system (UPS) is thought to be governed primarily by the recognition of specific motifs — degrons — present in substrate proteins. The ends of proteins — the N- and C-termini – have unique properties, and an important subset of protein–protein interactions involve the recognition of free termini. The first degrons to be discovered were located at the extreme N-terminus of proteins, a finding which initiated the study of the N-degron (formerly N-end rule) pathways, but only in the last few years has it emerged that a diverse set of C-degron pathways target analogous degron motifs located at the extreme C-terminus of proteins. In this minireview we summarise the N-degron and C-degron pathways currently known to operate in human cells, focussing primarily on those that have been discovered in recent years. In each case we describe the cellular machinery responsible for terminal degron recognition, and then consider some of the functional roles of terminal degron pathways. Altogether, a broad spectrum of E3 ubiquitin ligases mediate the recognition of a diverse array of terminal degron motifs; these degradative pathways have the potential to influence a wide variety of cellular functions.

Role of Apis cerana cerana N-terminal asparagine amidohydrolase (AccNtan1) in oxidative stress.

N-Terminal asparagine amidohydrolase is a component of the ubiquitin-dependent N-end rule pathway of protein degradation that has been implicated in a variety of physiological functions, including the sensing of heme, oxygen, nitric oxide, selective elimination of misfolded proteins and the repair of DNA. We identified the Apis cerana cerana N-terminal asparagine amidohydrolase (AccNtan1) gene from A. cerana cerana and investigated its role in oxidation resistance. Multiple sequence alignments and phylogenetic analysis revealed that N-terminal asparagine amidohydrolase is highly conserved in insect species. Quantitative real-time polymerase chain reaction analysis indicated that the expression levels of AccNtan1 were significantly lower in the wing, honey sac and abdomen than in other tissues and were significantly higher in early stages of development, including the larva, prepupa and pink-eyed pupa stages, than in later stages. We further observed that AccNtan1 expression was induced by several environmental stressors, including aberrant temperature, H2O2, UV, heavy metals and pesticides. Moreover, a bacteriostatic assay suggested that overexpression of AccNtan1 enhances the resistance of bacteria to oxidative stress. In addition, knockdown of AccNtan1 using RNA interference significantly affected the expression levels of most antioxidant genes and the activity levels of several antioxidant enzymes. Thus, we hypothesize that AccNtan1 plays important roles in environmental stress responses and antioxidative processes.

NatB regulates Rb mutant cell death and tumor growth by modulating EGFR/MAPK signaling through the N-end rule pathways.

Inactivation of the Rb tumor suppressor causes context-dependent increases in cell proliferation or cell death. In a genetic screen for factors that promoted Rb mutant cell death in Drosophila, we identified Psid, a regulatory subunit of N-terminal acetyltransferase B (NatB). We showed that NatB subunits were required for elevated EGFR/MAPK signaling and Rb mutant cell survival. We showed that NatB regulates the posttranscriptional levels of the highly conserved pathway components Grb2/Drk, MAPK, and PP2AC but not that of the less conserved Sprouty. Interestingly, NatB increased the levels of positive pathway components Grb2/Drk and MAPK while decreased the levels of negative pathway component PP2AC, which were mediated by the distinct N-end rule branch E3 ubiquitin ligases Ubr4 and Cnot4, respectively. These results suggest a novel mechanism by which NatB and N-end rule pathways modulate EGFR/MAPK signaling by inversely regulating the levels of multiple conserved positive and negative pathway components. As inactivation of Psid blocked EGFR signaling-dependent tumor growth, this study raises the possibility that NatB is potentially a novel therapeutic target for cancers dependent on deregulated EGFR/Ras signaling.