The Experts below are selected from a list of 2187 Experts worldwide ranked by ideXlab platform
Alexander Varshavsky - One of the best experts on this subject based on the ideXlab platform.
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Understanding the Pro/N-End Rule pathway
Nature Chemical Biology, 2018Co-Authors: David A. Dougan, Alexander VarshavskyAbstract:Regulated destruction of proteins underlies just about everything a cell does. A structural study of human Gid4, the N-recognin of the Pro/N-End Rule pathway that targets proteins through their N-terminal proline, illuminates the recognition of substrates by this proteolytic system.
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Control of Hsp90 chaperone and its clients by N-terminal acetylation and the N-End Rule pathway.
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Jang Hyun Oh, Ju-yeon Hyun, Alexander VarshavskyAbstract:We found that the heat shock protein 90 (Hsp90) chaperone system of the yeast Saccharomyces cerevisiae is greatly impaired in naa10Δ cells, which lack the NatA N α -terminal acetylase (Nt-acetylase) and therefore cannot N-terminally acetylate a majority of normally N-terminally acetylated proteins, including Hsp90 and most of its cochaperones. Chk1, a mitotic checkpoint kinase and a client of Hsp90, was degraded relatively slowly in wild-type cells but was rapidly destroyed in naa10Δ cells by the Arg/N-End Rule pathway, which recognized a C terminus-proximal degron of Chk1. Diverse proteins (in addition to Chk1) that are shown here to be targeted for degradation by the Arg/N-End Rule pathway in naa10Δ cells include Kar4, Tup1, Gpd1, Ste11, and also, remarkably, the main Hsp90 chaperone (Hsc82) itself. Protection of Chk1 by Hsp90 could be overridden not only by ablation of the NatA Nt-acetylase but also by overexpression of the Arg/N-End Rule pathway in wild-type cells. Split ubiquitin-binding assays detected interactions between Hsp90 and Chk1 in wild-type cells but not in naa10Δ cells. These and related results revealed a major role of Nt-acetylation in the Hsp90-mediated protein homeostasis, a strong up-regulation of the Arg/N-End Rule pathway in the absence of NatA, and showed that a number of Hsp90 clients are previously unknown substrates of the Arg/N-End Rule pathway.
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degradation of serotonin n acetyltransferase a circadian regulator by the n end Rule pathway
Journal of Biological Chemistry, 2016Co-Authors: Brandon Wadas, Cheol-sang Hwang, Jimo Borjigin, Zheping Huang, Jang Hyun Oh, Alexander VarshavskyAbstract:Serotonin N-acetyltransferase (AANAT) converts serotonin to N-acetylserotonin (NAS), a distinct biological regulator and the immediate precursor of melatonin, a circulating hormone that influences circadian processes, including sleep. N-terminal sequences of AANAT enzymes vary among vertebrates. Mechanisms that regulate the levels of AANAT are incompletely understood. Previous findings were consistent with the possibility that AANAT may be controlled through its degradation by the N-End Rule pathway. By expressing the rat and human AANATs and their mutants not only in mammalian cells but also in the yeast Saccharomyces cerevisiae, and by taking advantage of yeast genetics, we show here that two complementary forms of rat AANAT are targeted for degradation by two complementary branches of the N-End Rule pathway. Specifically, the Nα terminally acetylated (Nt-acetylated) Ac-AANAT is destroyed through the recognition of its Nt acetylated N terminal Met residue by the Ac/N-End Rule pathway, whereas the non Nt acetylated AANAT is targeted by the Arg/N end Rule pathway, which recognizes the unacetylated N-terminal Met-Leu sequence of rat AANAT. We also show, by constructing lysine to arginine mutants of rat AANAT, that its degradation is mediated by polyubiquitylation of its Lys residue(s). Human AANAT, whose N-terminal sequence differs from that of rodent AANATs, is longer lived than its rat counterpart, and appears to be refractory to degradation by the N-End Rule pathway. Together, these and related results indicate both a major involvement of the N-End Rule pathway in the control of rodent AANATs and substantial differences in the regulation of rodent and human AANATs that stem from differences in their N terminal sequences.
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Control of mammalian G protein signaling by N-terminal acetylation and the N-End Rule pathway
Science, 2015Co-Authors: Sang Eun Park, Alexander Varshavsky, Brandon Wadas, Ok Hee Seok, Cheol-sang HwangAbstract:Rgs2, a regulator of G proteins, lowers blood pressure by decreasing signaling through Gαq. Human patients expressing Met-Leu-Rgs2 (ML-Rgs2) or Met-Arg-Rgs2 (MR-Rgs2) are hypertensive relative to people expressing wild-type Met-Gln-Rgs2 (MQ-Rgs2). We found that wild-type MQ-Rgs2 and its mutant, MR-Rgs2, were destroyed by the Ac/N-End Rule pathway, which recognizes Nα-terminally acetylated (Nt-acetylated) proteins. The shortest-lived mutant, ML-Rgs2, was targeted by both the Ac/N-End Rule and Arg/N-End Rule pathways. The latter pathway recognizes unacetylated N-terminal residues. Thus, the Nt-acetylated Ac-MX-Rgs2 (X = Arg, Gln, Leu) proteins are specific substrates of the mammalian Ac/N-End Rule pathway. Furthermore, the Ac/N-degron of Ac-MQ-Rgs2 was conditional, and Teb4, an endoplasmic reticulum (ER) membrane-embedded ubiquitin ligase, was able to regulate G protein signaling by targeting Ac-MX-Rgs2 proteins for degradation through their Nα-terminal acetyl group.
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Calpain-generated natural protein fragments as short-lived substrates of the N-End Rule pathway
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Konstantin I Piatkov, Jang Hyun Oh, Alexander VarshavskyAbstract:Calpains are Ca2+-dependent intracellular proteases. We show here that calpain-generated natural C-terminal fragments of proteins that include G protein–coupled receptors, transmembrane ion channels, transcriptional regulators, apoptosis controllers, kinases, and phosphatases (Phe-GluN2a, Lys-Ica512, Arg-Ankrd2, Tyr-Grm1, Arg-Atp2b2, Glu-Bak, Arg-Igfbp2, Glu-IκBα, and Arg-c-Fos), are short-lived substrates of the Arg/N-End Rule pathway, which targets destabilizing N-terminal residues. We also found that the identity of a fragment’s N-terminal residue can change during evolution, but the residue’s destabilizing activity is virtually always retained, suggesting selection pressures that favor a short half-life of the calpain-generated fragment. It is also shown that a self-cleavage of a calpain can result in an N-End Rule substrate. Thus, the autoprocessing of calpains can control them by making active calpains short-lived. These and related results indicate that the Arg/N-End Rule pathway mediates the remodeling of oligomeric complexes by eliminating protein fragments that are produced in these complexes through cleavages by calpains or other nonprocessive proteases. We suggest that this capability of the Arg/N-End Rule pathway underlies a multitude of its previously known but mechanistically unclear functions.
Yong Tae Kwon - One of the best experts on this subject based on the ideXlab platform.
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The arginylation branch of the N-End Rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins.
Autophagy, 2016Co-Authors: Yanxialei Jiang, Won Hoon Choi, Hyunjoo Cha-molstad, Yong Tae KwonAbstract:ABSTRACTThe N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-End Rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-End Rule pathway), we identified that blocking the Arg/N-End Rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-End Rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the...
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Pharmacological Modulation of the N-End Rule Pathway and Its Therapeutic Implications
Trends in Pharmacological Sciences, 2015Co-Authors: Yanxialei Jiang, Yong Tae KwonAbstract:The N-End Rule pathway is a proteolytic system in which single N-terminal amino acids of short-lived substrates determine their metabolic half-lives. Substrates of this pathway have been implicated in the pathogenesis of many diseases, including malignancies, neurodegeneration, and cardiovascular disorders. This review provides a comprehensive overview of current knowledge about the mechanism and functions of the N-End Rule pathway. Pharmacological strategies for the modulation of target substrate degradation are also reviewed, with emphasis on their in vivo implications. Given the rapid advances in structural and biochemical understanding of the recognition components (N-recognins) of the N-End Rule pathway, small-molecule inhibitors and activating ligands of N-recognins emerge as therapeutic agents with novel mechanisms of action.
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The N-End Rule and retroviral infection: no effect on integrase
Virology Journal, 2013Co-Authors: Guney Boso, Takafumi Tasaki, Yong Tae Kwon, Nikunj V. SomiaAbstract:Background Integration of double stranded viral DNA is a key step in the retroviral life cycle. Virally encoded enzyme, integrase, plays a central role in this reaction. Mature forms of integrase of several retroviruses (i.e. HIV-1 and MLV) bear conserved destabilizing N-terminal residues of the N-End Rule pathway - a ubiquitin dependent proteolytic system in which the N-terminal residue of a protein determines its half life. Substrates of the N-End Rule pathway are recognized by E3 ubiquitin ligases called N-recognins. We have previously shown that the inactivation of three of these N-recognins, namely UBR1, UBR2 and UBR4 in mouse embryonic fibroblasts (MEFs) leads to increased stability of ectopically expressed HIV-1 integrase. These findings have prompted us to investigate the involvement of the N-End Rule pathway in the HIV-1 life cycle.
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The N-End Rule Pathway
Annual Review of Biochemistry, 2012Co-Authors: Takafumi Tasaki, Shashikanth M. Sriram, Kyong Soo Park, Yong Tae KwonAbstract: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...
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The N-End Rule Pathway
Annual Review of Biochemistry, 2012Co-Authors: Takafumi Tasaki, Shashikanth M. Sriram, Kyong Soo Park, Yong Tae KwonAbstract: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.
Shashikanth M. Sriram - One of the best experts on this subject based on the ideXlab platform.
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Characterization of mammalian N-degrons and development of heterovalent inhibitors of the N-End Rule pathway†
Chemical Science, 2013Co-Authors: Yanxialei Jiang, Shashikanth M. Sriram, Pritha Agarwalla, Subrata Pore, Adriana Zakrzewska, Rajkumar BanerjeeAbstract:The N-End Rule pathway relates the in vivo half-life of a protein with its N-terminal residue. Recent understanding of the molecular mechanism underlying N-degron recognition implies that the yeast N-degrons may not be identical to those of mammals. Here we re-evaluate the role of N-terminal amino acids as degradation determinants through an in vitro degradation assay and computational docking analysis. To take advantage of the distinct binding modes of type 1 and type 2 destabilizing residues, we developed and optimized heterovalent inhibitors of the N-End Rule pathway. These small-molecules effectively delayed the degradation of the physiological N-End Rule substrates in vitro and in living cells, including cardiomyocytes, suggesting that the heterovalent inhibitors could be applied to various cardiac diseases that originate from abnormal N-End Rule regulation.
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Development and Characterization of Monomeric N‑End Rule Inhibitors through In Vitro Model Substrates
Journal of Medicinal Chemistry, 2013Co-Authors: Shashikanth M. Sriram, Yanxialei Jiang, Rajkumar BanerjeeAbstract:In the N-End Rule pathway, a set of N-terminal amino acids, called N-degrons, are recognized and ubiquitinated by the UBR proteins. Here we examined various N-End Rule inhibitors to identify essential structural components of the system. Our study using in vitro biochemical assay indicated that the l-conformation and protonated α-amino group of the first residue were critical for N-degrons to properly interact with the UBR proteins. The monomeric molecules with minimum interacting motifs showed endopeptidase resistance and better inhibitory activities than traditional dipeptide inhibitors. Collectively, our study identifies a pharmacophore of N-End Rule inhibitors, which provides a structural platform to improve the efficiency and druggable properties of inhibitors. Considering that the N-End Rule has been implicated in many pathophysiological processes in cells, inhibitors of this pathway, such as p-chloroamphetamine, are potentially of clinical interest in a novel aspect of action mechanisms.
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The N-End Rule Pathway
Annual Review of Biochemistry, 2012Co-Authors: Takafumi Tasaki, Shashikanth M. Sriram, Kyong Soo Park, Yong Tae KwonAbstract: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...
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The N-End Rule Pathway
Annual Review of Biochemistry, 2012Co-Authors: Takafumi Tasaki, Shashikanth M. Sriram, Kyong Soo Park, Yong Tae KwonAbstract: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.
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The N-End Rule pathway: emerging functions and molecular principles of substrate recognition
Nature Reviews Molecular Cell Biology, 2011Co-Authors: Shashikanth M. Sriram, Yong Tae KwonAbstract:The N-End Rule defines the protein-destabilizing activity of a given amino-terminal residue following its post-translational modification. The N-End Rule pathway is emerging as a major cellular proteolytic system, and recent studies provide insights into its components, substrates and functions, as well as the structural basis of substrate recognition. 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. The N-End Rule defines the destabilizing activity of a given amino-terminal residue and its post-translational modification. Recognition components (N-recognins) of the N-End Rule pathway recognize destabilizing N-terminal amino acids as an essential element of specific N-terminal degrons (N-degrons). A functional N-degron is typically composed of a destabilizing N-terminal residue, an internal Lys residue (the site of polyubiquitylation) and an unstructured N-terminal extension. N-degrons can be generated through endoproteolytic cleavage of polypeptides, which exposes embedded N-degrons at the N termini of C-terminal fragments, or through the post-translational modification of pro-N-degrons, including their deamidation, oxidation, arginylation and acetylation. In mammals, N-recognins characterized by the UBR box bind N-degrons and subsequently induce ubiquitylation and proteasomal degradation, whereas N-recognins in bacteria mediate proteolysis without ubiquitin-like molecules. UBR1 (ubiquitin ligase N-recognin 1)-type N-recognins recognize type 1 and type 2 N-degrons through two distinct sites, the UBR box and the N-domain. The UBR box is a zinc-finger domain that binds a positively charged type 1 residue through a negatively charged, shallow groove. The N-domain appears to have evolutionarily originated from the bacterial N-recognin ClpS which binds a bulky hydrophobic type 2 residue through a deep hydrophobic pocket, within which the N-terminal side chain of the substrate is completely buried. In Saccharomyces cerevisiae , Ubr1 of the N-End Rule pathway and ubiquitin-fusion degradation 4 (Ufd4) of the UFD pathway form a complex and synergistically mediate ubiquitylation for both pathways. In complex with Ubr1, Ufd4 functions as an E4-like processivity-enhancing cofactor for Lys48-linked ubiquitylation by Ubr1 after Ubr1 recognizes a substrate. In S. cerevisiae , an acetylated N-terminal residue, including the retained initiator Met, can act as an N-degron, thereby functioning as an alternative signal to initiate the N-End Rule pathway. The Doa10 E3 ligase, in concert with the ubiquitin carrier 6 (Ubc6) or Ubc7 E2 enzymes, functions as a new type of N-recognin that mediates the polyubiquitylation of acetylated N-degrons.
Cheol-sang Hwang - One of the best experts on this subject based on the ideXlab platform.
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N-terminal acetylation and the N-End Rule pathway control degradation of the lipid droplet protein PLIN2.
Journal of Biological Chemistry, 2018Co-Authors: Nhung Thimy Truong, Sang Ki Park, Cheol-sang HwangAbstract:: 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.
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Control of protein degradation by N-terminal acetylation and the N-End Rule pathway.
Experimental and Molecular Medicine, 2018Co-Authors: Cheol-sang HwangAbstract:Nα-terminal acetylation (Nt-acetylation) occurs very frequently and is found in most proteins in eukaryotes. Despite the pervasiveness and universality of Nt-acetylation, its general functions in terms of physiological outcomes remain largely elusive. However, several recent studies have revealed that Nt-acetylation has a significant impact on protein stability, activity, folding patterns, cellular localization, etc. In addition, Nt-acetylation marks specific proteins for degradation by a branch of the N-End Rule pathway, a subset of the ubiquitin-mediated proteolytic system. The N-End Rule associates a protein’s in vivo half-life with its N-terminal residue or modifications on its N-terminus. This review provides a current understanding of intracellular proteolysis control by Nt-acetylation and the N-End Rule pathway. The addition of an acetyl group to amino acids at the start of a protein (known as the Nα-terminal end) is crucial for maintaining protein homeostasis and cellular health. Hwang and colleagues review the effects of Nα-terminal acetylation (Nt-acetylation) on protein function and stability. This modification occurs in over 50% of proteins in eukaryotic organisms and when mis-regulated can lead to cancer, hypertension, neurodegeneration etc. Nt-acetylation not only targets proteins for degradation through a specific signaling pathway, but also regulates protein folding, cellular localization and activity. This seemingly irreversible modification can also protect proteins against other mechanisms of degradation and represents a potential therapeutic target. Inhibiting Nt-acetylation-dependent protein interactions could be a useful strategy for regulating protein stability.
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degradation of serotonin n acetyltransferase a circadian regulator by the n end Rule pathway
Journal of Biological Chemistry, 2016Co-Authors: Brandon Wadas, Cheol-sang Hwang, Jimo Borjigin, Zheping Huang, Jang Hyun Oh, Alexander VarshavskyAbstract:Serotonin N-acetyltransferase (AANAT) converts serotonin to N-acetylserotonin (NAS), a distinct biological regulator and the immediate precursor of melatonin, a circulating hormone that influences circadian processes, including sleep. N-terminal sequences of AANAT enzymes vary among vertebrates. Mechanisms that regulate the levels of AANAT are incompletely understood. Previous findings were consistent with the possibility that AANAT may be controlled through its degradation by the N-End Rule pathway. By expressing the rat and human AANATs and their mutants not only in mammalian cells but also in the yeast Saccharomyces cerevisiae, and by taking advantage of yeast genetics, we show here that two complementary forms of rat AANAT are targeted for degradation by two complementary branches of the N-End Rule pathway. Specifically, the Nα terminally acetylated (Nt-acetylated) Ac-AANAT is destroyed through the recognition of its Nt acetylated N terminal Met residue by the Ac/N-End Rule pathway, whereas the non Nt acetylated AANAT is targeted by the Arg/N end Rule pathway, which recognizes the unacetylated N-terminal Met-Leu sequence of rat AANAT. We also show, by constructing lysine to arginine mutants of rat AANAT, that its degradation is mediated by polyubiquitylation of its Lys residue(s). Human AANAT, whose N-terminal sequence differs from that of rodent AANATs, is longer lived than its rat counterpart, and appears to be refractory to degradation by the N-End Rule pathway. Together, these and related results indicate both a major involvement of the N-End Rule pathway in the control of rodent AANATs and substantial differences in the regulation of rodent and human AANATs that stem from differences in their N terminal sequences.
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N-Terminal Acetylation-Targeted N-End Rule Proteolytic System: The Ac/N-End Rule Pathway
Molecules and Cells, 2016Co-Authors: Cheol-sang HwangAbstract:Although Nα-terminal acetylation (Nt-acetylation) is a pervasive protein modification in eukaryotes, its general functions in a majority of proteins are poorly understood. In 2010, it was discovered that Nt-acetylation creates a specific protein degradation signal that is targeted by a new class of the N-End Rule proteolytic system, called the Ac/N-End Rule pathway. Here, we review recent advances in our understanding of the mechanism and biological functions of the Ac/N-End Rule pathway, and its crosstalk with the Arg/N-End Rule pathway (the classical N-End Rule pathway).
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Control of mammalian G protein signaling by N-terminal acetylation and the N-End Rule pathway
Science, 2015Co-Authors: Sang Eun Park, Alexander Varshavsky, Brandon Wadas, Ok Hee Seok, Cheol-sang HwangAbstract:Rgs2, a regulator of G proteins, lowers blood pressure by decreasing signaling through Gαq. Human patients expressing Met-Leu-Rgs2 (ML-Rgs2) or Met-Arg-Rgs2 (MR-Rgs2) are hypertensive relative to people expressing wild-type Met-Gln-Rgs2 (MQ-Rgs2). We found that wild-type MQ-Rgs2 and its mutant, MR-Rgs2, were destroyed by the Ac/N-End Rule pathway, which recognizes Nα-terminally acetylated (Nt-acetylated) proteins. The shortest-lived mutant, ML-Rgs2, was targeted by both the Ac/N-End Rule and Arg/N-End Rule pathways. The latter pathway recognizes unacetylated N-terminal residues. Thus, the Nt-acetylated Ac-MX-Rgs2 (X = Arg, Gln, Leu) proteins are specific substrates of the mammalian Ac/N-End Rule pathway. Furthermore, the Ac/N-degron of Ac-MQ-Rgs2 was conditional, and Teb4, an endoplasmic reticulum (ER) membrane-embedded ubiquitin ligase, was able to regulate G protein signaling by targeting Ac-MX-Rgs2 proteins for degradation through their Nα-terminal acetyl group.
Ilia V Davydov - One of the best experts on this subject based on the ideXlab platform.
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rgs4 and rgs5 are in vivo substrates of the n end Rule pathway
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Takafumi Tasaki, Kayoko Moroi, Jee Young An, Sadao Kimura, Ilia V Davydov, Yong Tae KwonAbstract:The ATE1-encoded Arg-transferase mediates conjugation of Arg to N-terminal Asp, Glu, and Cys of certain eukaryotic proteins, yielding N-terminal Arg that can act as a degradation signal for the ubiquitin-dependent N-End Rule pathway. We have previously shown that mouse ATE1–/– embryos die with defects in heart development and angiogenesis. Here, we report that the ATE1 Arg-transferase mediates the in vivo degradation of RGS4 and RGS5, which are negative regulators of specific G proteins whose functions include cardiac growth and angiogenesis. The proteolysis of these regulators of G protein signaling (RGS) proteins was perturbed either by hypoxia or in cells lacking ubiquitin ligases UBR1 and/or UBR2. Mutant RGS proteins in which the conserved Cys-2 residue could not become N-terminal were long-lived in vivo. We propose a model in which the sequential modifications of RGS4, RGS5, and RGS16 (N-terminal exposure of their Cys-2, its oxidation, and subsequent arginylation) act as a licensing mechanism in response to extracellular and intracellular signals before the targeting for proteolysis by UBR1 and UBR2. We also show that ATE1–/– embryos are impaired in the activation of extracellular signal-regulated kinase mitogen-activated protein kinases and in the expression of G protein-induced downstream effectors such as Jun, cyclin D1, and β-myosin heavy chain. These results establish RGS4 and RGS5 as in vivo substrates of the mammalian N-End Rule pathway and also suggest that the O2-ATE1-UBR1/UBR2 proteolytic circuit plays a role in RGS-regulated G protein signaling in the cardiovascular system.
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Female lethality and apoptosis of spermatocytes in mice lacking the UBR2 ubiquitin ligase of the N-End Rule pathway.
Molecular and Cellular Biology, 2003Co-Authors: Yong Tae Kwon, Takafumi Tasaki, Jee Young An, Ilia V Davydov, Jun Sheng, Alexander VarshavskyAbstract:Substrates of the ubiquitin-dependent N-End Rule pathway include proteins with destabilizing N-terminal residues. UBR1-/- mice, which lacked the pathway's ubiquitin ligase E3α, were viable and retained the N-End Rule pathway. The present work describes the identification and analysis of mouse UBR2, a homolog of UBR1. We demonstrate that the substrate-binding properties of UBR2 are highly similar to those of UBR1, identifying UBR2 as the second E3 of the mammalian N-End Rule pathway. UBR2-/- mouse strains were constructed, and their viability was found to be dependent on both gender and genetic background. In the strain 129 (inbred) background, the UBR2-/- genotype was lethal to most embryos of either gender. In the 129/B6 (mixed) background, most UBR2-/- females died as embryos, whereas UBR2-/- males were viable but infertile, owing to the postnatal degeneration of the testes. The gross architecture of UBR2-/- testes was normal and spermatogonia were intact as well, but UBR2-/- spermatocytes were arrested between leptotene/zygotene and pachytene and died through apoptosis. A conspicuous defect of UBR2-/- spermatocytes was the absence of intact synaptonemal complexes. We conclude that the UBR2 ubiquitin ligase and, hence, the N-End Rule pathway are required for male meiosis and spermatogenesis and for an essential aspect of female embryonic development.
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rgs4 is arginylated and degraded by the n end Rule pathway in vitro
Journal of Biological Chemistry, 2000Co-Authors: Ilia V Davydov, Alexander VarshavskyAbstract:Abstract The N-End Rule relates the in vivohalf-life of a protein to the identity of its N-terminal residue. We used an expression-cloning screen to search for mouse proteins that are degraded by the ubiquitin/proteasome-dependent N-End Rule pathway in a reticulocyte lysate. One substrate thus identified was RGS4, a member of the RGS family of GTPase-activating proteins that down-regulate specific G proteins. A determinant of the RGS4 degradation signal (degron) was located at the N terminus of RGS4, because converting cysteine 2 to either glycine, alanine, or valine completely stabilized RGS4. Radiochemical sequencing indicated that the N-terminal methionine of the lysate-produced RGS4 was replaced with arginine. Since N-terminal arginine is a destabilizing residue not encoded by RGS4 mRNA, we conclude that the degron of RGS4 is generated through the removal of N-terminal methionine and enzymatic arginylation of the resulting N-terminal cysteine. RGS16, another member of the RGS family, was also found to be an N-End Rule substrate. RGS4 that was transiently expressed in mouse L cells was short-lived in these cells. However, the targeting of RGS4 for degradation in this in vivo setting involved primarily another degron, because N-terminal variants of RGS4 that were stable in reticulocyte lysate remained unstable in L cells.
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The N-End Rule Pathway inXenopusEgg Extracts
Archives of Biochemistry and Biophysics, 1998Co-Authors: Ilia V Davydov, Debabrata Patra, Alexander VarshavskyAbstract:Abstract Ubiquitin-dependent degradation of intracellular proteins underlies a multitude of biological processes, including the cell cycle, cell differentiation, and responses to stress. One ubiquitin-dependent proteolytic system is the N-End Rule pathway, whose targets include proteins that bear destabilizing N-terminal residues. This pathway, which has been characterized only in somatic cells, is shown here to be present also in germ line cells such as the eggs of the amphibian Xenopus laevis. We demonstrate that the set of destabilizing residues in the N-End Rule pathway of Xenopus eggs is similar, if not identical, to that of somatic cells such as mammalian reticulocytes and fibroblasts. It is also shown that the degradation of engineered N-End Rule substrates in egg extracts can be strongly and selectively inhibited by dipeptides bearing destabilizing N-terminal residues. This result allowed us to ask whether selective inhibition of the N-End Rule pathway in egg extracts influences the apoptosis-like changes that are observed in these extracts. A dipeptide bearing a bulky hydrophobic (type 2) destabilizing N-terminal residue was found to delay the apoptotic changes in egg extracts, whereas dipeptides bearing basic (type 1) destabilizing N-terminal residues had no effect. High activity of the N-End Rule pathway in egg extracts provides an alternative to reticulocyte extracts for the in vitro analyses of this pathway.
