N-end Rule Pathway Research Articles

Engineering ClpS for selective and enhanced N-terminal amino acid binding.

One of the central challenges in the development of single-molecule protein sequencing technologies is achieving high-fidelity sequential recognition and detection of specific amino acids that comprise the peptide sequence. An approach towards achieving this goal is to leverage naturally occurring proteins that function through recognition of amino (N)-terminal amino acids (NAAs). One such protein, the N-end rule pathway adaptor protein ClpS, natively recognizes NAAs on a peptide chain. The native ClpS protein has a high specificity albeit modest affinity for the amino acid Phe at the N-terminus but also recognizes the residues Trp, Tyr, and Leu at the N-terminal position. Here, we employed directed evolution methods to select for ClpS variants with enhanced affinity and selectivity for two NAAs (Phe and Trp). Using this approach, we identified two promising variants of the Agrobacterium tumefaciens ClpS protein with native residues 34-36 ProArgGlu mutated to ProMetSer and CysProSer. In vitro surface binding assays indicate that the ProMetSer variant has enhanced affinity for Phe at the N-terminus with sevenfold tighter binding relative to wild-type ClpS, and that the CysProSer variant binds selectively to Trp over Phe at the N-terminus while having a greater affinity for both Trp and Phe. Taken together, this work demonstrates the utility of engineering ClpS to make it more effective for potential use in peptide sequencing applications.

N-degron and C-degron pathways of protein degradation

This perspective is partly review and partly proposal. N-degrons and C-degrons are degradation signals whose main determinants are, respectively, the N-terminal and C-terminal residues of cellular proteins. N-degrons and C-degrons include, to varying extents, adjoining sequence motifs, and also internal lysine residues that function as polyubiquitylation sites. Discovered in 1986, N-degrons were the first degradation signals in short-lived proteins. A particularly large set of C-degrons was discovered in 2018. We describe multifunctional proteolytic systems that target N-degrons and C-degrons. We also propose to denote these systems as “N-degron pathways” and “C-degron pathways.” The former notation replaces the earlier name “N-end rule pathways.” The term “N-end rule” was introduced 33 years ago, when only some N-terminal residues were thought to be destabilizing. However, studies over the last three decades have shown that all 20 amino acids of the genetic code can act, in cognate sequence contexts, as destabilizing N-terminal residues. Advantages of the proposed terms include their brevity and semantic uniformity for N-degrons and C-degrons. In addition to being topologically analogous, N-degrons and C-degrons are related functionally. A proteolytic cleavage of a subunit in a multisubunit complex can create, at the same time, an N-degron (in a C-terminal fragment) and a spatially adjacent C-degron (in an N-terminal fragment). Consequently, both fragments of a subunit can be selectively destroyed through attacks by the N-degron and C-degron pathways.

N-terminal acetylation and the N-end rule pathway control degradation of the lipid droplet protein PLIN2

Perilipin 2 (PLIN2) is a major lipid droplet (LD)-associated protein that regulates intracellular lipid homeostasis and LD formation. Under lipid-deprived conditions, the LD-unbound (free) form of PLIN2 is eliminated in the cytosol by an as yet unknown ubiquitin (Ub)-proteasome pathway that is associated with the N-terminal or near N-terminal residues of the protein. Here, using HeLa, HEK293T, and HepG2 human cell lines, cycloheximide chase, in vivo ubiquitylation, split-Ub yeast two-hybrid, and chemical cross-linking-based reciprocal co-immunoprecipitation assays, we found that TEB4 (MARCH6), an E3 Ub ligase and recognition component of the Ac/N-end rule pathway, directly targets the N-terminal acetyl moiety of Nα-terminally acetylated PLIN2 for its polyubiquitylation and degradation by the 26S proteasome. We also show that the TEB4-mediated Ac/N-end rule pathway reduces intracellular LD accumulation by degrading PLIN2. Collectively, these findings identify PLIN2 as a substrate of the Ac/N-end rule pathway and indicate a previously unappreciated role of the Ac/N-end rule pathway in LD metabolism.

Regulation of NLR stability in plant immunity

Plant nucleotide binding domain and leucine-rich repeat (NLR) receptors recognize pathogen effectors directly or indirectly and mediate innate immune responses. NLR-mediated immunity also has direct impacts on plant growth and development, as well as yield and survival. The levels of NLR proteins are therefore intricately controlled in plants to balance defense responses and other processes. In recent years, the ubiquitination-26S proteasome system and the HSP90 chaperones have emerged as having key functions in the regulation of NLR stability. The N-end rule pathway of protein degradation is also directly linked to NLR stability. Recent progress in the regulation of NLR stability and turnover is summarized here, focusing on the key components and pathways.

Oxygen-dependent proteolysis regulates the stability of angiosperm polycomb repressive complex 2 subunit VERNALIZATION\xa02

The polycomb repressive complex 2 (PRC2) regulates epigenetic gene repression in eukaryotes. Mechanisms controlling its developmental specificity and signal-responsiveness are poorly understood. Here, we identify an oxygen-sensitive N-terminal (N-) degron in the plant PRC2 subunit VERNALIZATION(VRN) 2, a homolog of animal Su(z)12, that promotes its degradation via the N-end rule pathway. We provide evidence that this N-degron arose early during angiosperm evolution via gene duplication and N-terminal truncation, facilitating expansion of PRC2 function in flowering plants. We show that proteolysis via the N-end rule pathway prevents ectopic VRN2 accumulation, and that hypoxia and long-term cold exposure lead to increased VRN2 abundance, which we propose may be due to inhibition of VRN2 turnover via its N-degron. Furthermore, we identify an overlap in the transcriptional responses to hypoxia and prolonged cold, and show that VRN2 promotes tolerance to hypoxia. Our work reveals a mechanism for post-translational regulation of VRN2 stability that could potentially link environmental inputs to the epigenetic control of plant development.

Revisiting the Role of Ethylene and N-End Rule Pathway on Chilling-Induced Dormancy Release in Arabidopsis Seeds.

Dormant Arabidopsis (Arabidopsis thaliana) seeds do not germinate easily at temperatures higher than 10–15 °C. Using mutants affected in ethylene signaling (etr1, ein2 and ein4) and in the N-end-rule pathway of the proteolysis (prt6 and ate1-ate2) we have investigated the effects of cold and ethylene on dormancy alleviation. Ethylene (10–100 ppm) and 2–4 days chilling (4 °C) strongly stimulate the germination of wild type (Col-0) seeds at 25 °C. Two to four days of chilling promote the germination at 25 °C of all the mutants suggesting that release of dormancy by cold did not require ethylene and did not require the N-end-rule pathway. One mutant (etr1) that did not respond to ethylene did not respond to GA3 either. Mutants affected in the N-end rule (prt6 and ate1-ate2) did not respond to ethylene indicating that also this pathway is required for dormancy alleviation by ethylene; they germinated after chilling and in the presence of GA3. Cold can activate the ethylene signaling pathway since it induced an accumulation of ETR1, EINI4, and EIN2 transcripts, the expression of which was not affected by ethylene and GA3. Both cold followed by 10 h at 25 °C and ethylene downregulated the expression of PRT6, ATE1, ATE2, and of ABI5 involved in ABA signaling as compared to dormant seeds incubated at 25 °C. In opposite, the expression of RGA, GAI, and RGL2 encoding three DELLAs was induced at 4 °C but downregulated in the presence of ethylene.

Formyl-methionine as an N-degron of a eukaryotic N-end rule pathway.

In bacteria, nascent proteins bear the pretranslationally generated N-terminal (Nt) formyl-methionine (fMet) residue. Nt-fMet of bacterial proteins is a degradation signal, termed fMet/N-degron. By contrast, proteins synthesized by cytosolic ribosomes of eukaryotes were presumed to bear unformylated Nt-Met. Here we found that the yeast formyltransferase Fmt1, although imported into mitochondria, could also produce Nt-formylated proteins in the cytosol. Nt-formylated proteins were strongly up-regulated in stationary phase or upon starvation for specific amino acids. This up-regulation strictly required the Gcn2 kinase, which phosphorylates Fmt1 and mediates its retention in the cytosol. We also found that the Nt-fMet residues of Nt-formylated proteins act as fMet/N-degrons and identified the Psh1 ubiquitin ligase as the recognition component of the eukaryotic fMet/N-end rule pathway, which destroys Nt-formylated proteins.

Regulating Apoptosis by Degradation: The N-End Rule-Mediated Regulation of Apoptotic Proteolytic Fragments in Mammalian Cells.

A pivotal hallmark of some cancer cells is the evasion of apoptotic cell death. Importantly, the initiation of apoptosis often results in the activation of caspases, which, in turn, culminates in the generation of proteolytically-activated protein fragments with potentially new or altered roles. Recent investigations have revealed that the activity of a significant number of the protease-generated, activated, pro-apoptotic protein fragments can be curbed via their selective degradation by the N-end rule degradation pathways. Of note, previous work revealed that several proteolytically-generated, pro-apoptotic fragments are unstable in cells, as their destabilizing N-termini target them for proteasomal degradation via the N-end rule degradation pathways. Remarkably, previous studies also showed that the proteolytically-generated anti-apoptotic Lyn kinase protein fragment is targeted for degradation by the UBR1/UBR2 E3 ubiquitin ligases of the N-end rule pathway in chronic myeloid leukemia cells. Crucially, the degradation of cleaved fragment of Lyn by the N-end rule counters imatinib resistance in these cells, implicating a possible linkage between the N-end rule degradation pathway and imatinib resistance. Herein, we highlight recent studies on the role of the N-end rule proteolytic pathways in regulating apoptosis in mammalian cells, and also discuss some possible future directions with respect to apoptotic proteolysis signaling.

Insights on a putative aminoacyl-tRNA-protein transferase of Leishmania major.

The N-end rule pathway leads to regulated proteolysis as an adaptive response to external stress and is ubiquitous from bacteria to mammals. In this study, we investigated a gene coding for a putative core enzyme of this post-translational regulatory pathway in Leishmania major, which may be crucial during cytodifferentiation and the environment adaptive responses of the parasite. Leucyl, phenylalanyl-tRNA protein transferase and arginyl-tRNA protein transferase are key components of this pathway in E. coli and eukaryotes, respectively. They catalyze the specific conjugation of leucine, phenylalanine or arginine to proteins containing exposed N-terminal amino acid residues, which are recognized by the machinery for the targeted proteolysis. Here, we characterized a conserved hypothetical protein coded by the LmjF.21.0725 gene in L. major. In silico analysis suggests that the LmjF.21.0725 protein is highly conserved among species of Leishmania and might belong to the Acyl CoA-N-acyltransferases (NAT) superfamily of proteins. Immunofluorescence cell imaging indicates that the cytosolic localization of the studied protein and the endogenous levels of the protein in promastigotes are barely detectable by western blotting assay. The knockout of the two alleles of LmjF.21.0725 by homologous recombination was only possible in the heterozygous transfectant expressing LmjF.21.0725 as a transgene from a plasmid. Moreover, the kinetics of loss of the plasmid in the absence of drug pressure suggests that maintenance of the gene is essential for promastigote survival. Here, evidence is provided that this putative aminoacyl tRNA-protein transferase is essential for parasite survival. The enzyme activity and corresponding post-translational regulatory pathway are yet to be investigated.

The N-recognin UBR4 of the N-end rule pathway is targeted to and required for the biogenesis of the early endosome.

The N-end rule pathway is a proteolytic system in which single N-terminal residues of proteins act as N-degrons. These degrons are recognized by N-recognins, facilitating substrate degradation via the ubiquitin (Ub) proteasome system (UPS) or autophagy. We have previously identified a set of N-recognins [UBR1, UBR2, UBR4 (also known as p600) and UBR5 (also known as EDD)] that bind N-degrons through their UBR boxes to promote proteolysis by the proteasome. Here, we show that the 570 kDa N-recognin UBR4 is associated with maturing endosomes through an interaction with Ca2+-bound calmodulin. The endosomal recruitment of UBR4 is essential for the biogenesis of early endosomes (EEs) and endosome-related processes, such as the trafficking of endocytosed protein cargos and degradation of extracellular cargos by endosomal hydrolases. In mouse embryos, UBR4 marks and plays a role in the endosome-lysosome pathway that mediates the heterophagic proteolysis of endocytosed maternal proteins into amino acids. By screening 9591 drugs through the DrugBank database, we identify picolinic acid as a putative ligand for UBR4 that inhibits the biogenesis of EEs. Our results suggest that UBR4 is an essential modulator in the endosome-lysosome system.This article has an associated First Person interview with the first author of the paper.

First person – Sung Tae Kim

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Sung Tae Kim is the first author on ‘The N-recognin UBR4 of the N-end rule pathway is targeted to and required for the biogenesis of the early endosome’, published in Journal of Cell Science. Sung Tae Kim is a PhD student in the lab of Dr Yong Tae Kwon at University of Pittsburgh, Pittsburgh, USA, investigating the function of the N-end rule pathway in the ubiquitin-proteasome system and autophagy.

The N-recognin UBR4 of the N-end rule pathway is required for neurogenesis and homeostasis of cell surface proteins.

The N-end rule pathway is a proteolytic system in which single N-terminal amino acids of proteins act as a class of degrons (N-degrons) that determine the half-lives of proteins. We have previously identified a family of mammals N-recognins (termed UBR1, UBR2, UBR4/p600, and UBR5/EDD) whose conserved UBR boxes bind N-degrons to facilitate substrate ubiquitination and proteasomal degradation via the ubiquitin-proteasome system (UPS). Amongst these N-recognins, UBR1 and UBR2 mediate ubiquitination and proteolysis of short-lived regulators and misfolded proteins. Here, we characterized the null phenotypes of UBR4-deficient mice in which the UBR box of UBR4 was deleted. We show that the mutant mice die around embryonic days 9.5–10.5 (E9.5–E10.5) associated with abnormalities in various developmental processes such as neurogenesis and cardiovascular development. These developmental defects are significantly attributed to the inability to maintain cell integrity and adhesion, which significantly correlates to the severity of null phenotypes. UBR4-loss induces the depletion of many, but not all, proteins from the plasma membrane, suggesting that UBR4 is involved in proteome-wide turnover of cell surface proteins. Indeed, UBR4 is associated with and required to generate the multivesicular body (MVB) which transiently store endocytosed cell surface proteins before their targeting to autophagosomes and subsequently lysosomes. Our results suggest that the N-recognin UBR4 plays a role in the homeostasis of cell surface proteins and, thus, cell adhesion and integrity.

Distinct branches of the N-end rule pathway modulate the plant immune response.

The N-end rule pathway is a highly conserved constituent of the ubiquitin proteasome system, yet little is known about its biological roles. Here we explored the role of the N-end rule pathway in the plant immune response. We investigated the genetic influences of components of the pathway and known protein substrates on physiological, biochemical and metabolic responses to pathogen infection. We show that the glutamine (Gln) deamidation and cysteine (Cys) oxidation branches are both components of the plant immune system, through the E3 ligase PROTEOLYSIS (PRT)6. In Arabidopsis thaliana Gln-specific amino-terminal (Nt)-amidase (NTAQ1) controls the expression of specific defence-response genes, activates the synthesis pathway for the phytoalexin camalexin and influences basal resistance to the hemibiotroph pathogen Pseudomonas syringae pv tomato (Pst). The Nt-Cys ETHYLENE RESPONSE FACTOR VII transcription factor substrates enhance pathogen-induced stomatal closure. Transgenic barley with reduced HvPRT6 expression showed enhanced resistance to Ps. japonica and Blumeria graminis f. sp. hordei, indicating a conserved role of the pathway. We propose that that separate branches of the N-end rule pathway act as distinct components of the plant immune response in flowering plants.

Insights into degradation mechanism of N-end rule substrates by p62/SQSTM1 autophagy adapter

p62/SQSTM1 is the key autophagy adapter protein and the hub of multi-cellular signaling. It was recently reported that autophagy and N-end rule pathways are linked via p62. However, the exact recognition mode of degrading substrates and regulation of p62 in the autophagic pathway remain unknown. Here, we present the complex structures between the ZZ-domain of p62 and various type-1 and type-2 N-degrons. The binding mode employed in the interaction of the ZZ-domain with N-degrons differs from that employed by classic N-recognins. It was also determined that oligomerization via the PB1 domain can control functional affinity to the R-BiP substrate. Unexpectedly, we found that self-oligomerization and disassembly of p62 are pH-dependent. These findings broaden our understanding of the functional repertoire of the N-end rule pathway and provide an insight into the regulation of p62 during the autophagic pathway.

The plant cysteine oxidases from Arabidopsis thaliana are kinetically tailored to act as oxygen sensors

Group VII ethylene response factors (ERF-VIIs) regulate transcriptional adaptation to flooding-induced hypoxia in plants. ERF-VII stability is controlled in an O2-dependent manner by the Cys/Arg branch of the N-end rule pathway whereby oxidation of a conserved N-terminal cysteine residue initiates target degradation. This oxidation is catalyzed by plant cysteine oxidases (PCOs), which use O2 as cosubstrate to generate Cys-sulfinic acid. The PCOs directly link O2 availability to ERF-VII stability and anaerobic adaptation, leading to the suggestion that they act as plant O2 sensors. However, their ability to respond to fluctuations in O2 concentration has not been established. Here, we investigated the steady-state kinetics of Arabidopsis thaliana PCOs 1–5 to ascertain whether their activities are sensitive to O2 levels. We found that the most catalytically competent isoform is AtPCO4, both in terms of responding to O2 and oxidizing AtRAP2.2/2,12 (two of the most prominent ERF-VIIs responsible for promoting the hypoxic response), which suggests that AtPCO4 plays a central role in ERF-VII regulation. Furthermore, we found that AtPCO activity is susceptible to decreases in pH and that the hypoxia-inducible AtPCOs 1/2 and the noninducible AtPCOs 4/5 have discrete AtERF-VII substrate preferences. Pertinently, the AtPCOs had Km(O2)app values in a physiologically relevant range, which should enable them to sensitively react to changes in O2 availability. This work validates an O2-sensing role for the PCOs and suggests that differences in expression pattern, ERF-VII selectivity, and catalytic capability may enable the different isoforms to have distinct biological functions. Individual PCOs could therefore be targeted to manipulate ERF-VII levels and improve stress tolerance in plants.

N-terminal acetylation: an essential protein modification emerges as an important regulator of stress responses.

N-terminal acetylation (NTA) is a prevalent protein modification in eukaryotes. The majority of proteins are acetylated at their N-terminus in a co-translational manner by ribosome-associated N-terminal acetyltransferases (NATs). However, the recent discovery of Golgi membrane-localized NATs in metazoa, and plastid-localized NATs in plants challenged the dogma of static, co-translational imprinting of the proteome by NTA. Indeed, NTA by the cytosolic NatA is highly dynamic and under hormonal control in plants. Such active control has not been evidenced yet in other eukaryotes and might be an adaptation to the sessile lifestyle of plants forcing them to cope with diverse environmental challenges. The function of NTAs for individual proteins is distinct and yet unpredictable. In yeast and humans, NTA has been shown to affect protein-protein interactions, subcellular localization, folding, aggregation, or degradation of a handful of proteins. In particular, the impact of NTA on the protein turnover is documented by diverse examples in yeast. Consequently, NTA was recently dicovered to be a degradation signal in a distinct branch of the N-end rule pathway, ubiquitin-mediated proteolysis. In this review, we summarize the current knowledge on the NAT machinery in higher plants and discuss the potential function of NTA during biotic and abiotic stresses.

Comparative Transcriptome Analysis of Waterlogging-Sensitive and Waterlogging-Tolerant Chrysanthemum morifolium Cultivars under Waterlogging Stress and Reoxygenation Conditions.

Waterlogging stress is among the most severe abiotic stressors in the breeding and the production of Chrysanthemum morifolium. However, the mechanism underlying the response to waterlogging and post-waterlogging reoxygenation in C. morifolium remains unknown. In this study, we compared the differences between the transcriptomes of two chrysanthemum cultivars, i.e., the waterlogging-tolerant cultivar “Nannongxuefeng” and the waterlogging-sensitive cultivar “Qinglu”, by performing RNA-seq to elucidate the possible mechanism of waterlogging and reoxygenation in C. morifolium. “Nannongxuefeng” had a higher ethylene production under the waterlogging and reoxygenation conditions. Furthermore, the expression of transcription factors and genes that are involved in the hormone response, N-end rule pathway and ROS signaling significantly differed between the two cultivars. “Nannongxuefeng” and “Qinglu” significantly differed in their response to waterlogging and reoxygenation, providing a deeper understanding of the mechanism underlying the response to waterlogging and guidance for the breeding of C. morifolium.

C-Terminal End-Directed Protein Elimination by CRL2 Ubiquitin Ligases

The proteolysis-assisted protein quality control system guards the proteome from potentially detrimental aberrant proteins. How miscellaneous defective proteins are specifically eliminated and which molecular characteristics direct them for removal are fundamental questions. We reveal a mechanism, DesCEND (destruction via C-end degrons), by which CRL2 ubiquitin ligase uses interchangeable substrate receptors to recognize the unusual C termini of abnormal proteins (i.e., C-end degrons). C-end degrons are mostly less than ten residues in length and comprise a few indispensable residues along with some rather degenerate ones. The C-terminal end position is essential for C-end degron function. Truncated selenoproteins generated by translationerrors and the USP1 N-terminal fragment frompost-translational cleavage are eliminated by DesCEND. DesCEND also targets full-length proteins with naturally occurring C-end degrons. The C-end degron in DesCEND echoes the N-end degron in the N-end rule pathway, highlighting the dominance of protein "ends" as indicators for protein elimination.

Mapping Degradation Signals and Pathways in a Eukaryotic N-terminome

Most eukaryotic proteins are N-terminally acetylated. This modification can be recognized as a signal for selective protein degradation (degron) by the N-end rule pathways. However, the prevalence and specificity of such degrons in the proteome are unclear. Here, by systematically examining how protein turnover is affected by N-terminal sequences, we perform a comprehensive survey of degrons in the yeast N-terminome. We find that approximately 26% of nascent protein N termini encode cryptic degrons. These degrons exhibit high hydrophobicity and are frequently recognized by the E3 ubiquitin ligase Doa10, suggesting a role in protein quality control. In contrast, N-terminal acetylation rarely functions as a degron. Surprisingly, we identify two pathways where N-terminal acetylation has the opposite function and blocks protein degradation through the E3 ubiquitin ligase Ubr1. Our analysis highlights the complexity of N-terminal degrons and argues that hydrophobicity, not N-terminal acetylation, is the predominant feature of N-terminal degrons in nascent proteins.

Molecular basis of GID4-mediated recognition of degrons for the Pro/N-end rule pathway.

The N-end rule pathway senses the N-terminal destabilizing residues of degradation substrates for the ubiquitin-proteasome system, whose integrity shields against various human syndromes including cancer and cardiovascular diseases. GID4, a subunit of the ubiquitin ligase GID complex, has been recently identified as the N-recognin of the new branch of the N-end rule pathway responsible for recognizing substrates bearing N-terminal proline residues (Pro/N-degrons). However, the molecular mechanism of GID4-mediated Pro/N-degron recognition remains largely unexplored. Here, we report the first crystal structures of human GID4 alone and in complex with various Pro/N-degrons. Our complex crystal structures, together with biophysical analyses, delineate the GID4-mediated Pro/N-degron recognition mechanism and substrate selection criteria for the Pro/N-end rule pathway. These mechanistic data on the Pro/N-recognin activity of GID4 will serve as a foundation to facilitate the identification of authentic physiological substrates as well as the development of inhibitors of therapeutic values for the Pro/N-end rule pathway.