Rule Pathway Of Protein Degradation Research Articles

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.

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.

Structural basis for dual specificity of yeast N-terminal amidase in the N-end rule pathway

The first step of the hierarchically organized Arg/N-end rule pathway of protein degradation is deamidation of the N-terminal glutamine and asparagine residues of substrate proteins to glutamate and aspartate, respectively. These reactions are catalyzed by the N-terminal amidase (Nt-amidase) Nta1 in fungi such as Saccharomyces cerevisiae, and by the glutamine-specific Ntaq1 and asparagine-specific Ntan1 Nt-amidases in mammals. To investigate the dual specificity of yeast Nta1 (yNta1) and the importance of second-position residues in Asn/Gln-bearing N-terminal degradation signals (N-degrons), we determined crystal structures of yNta1 in the apo state and in complex with various N-degron peptides. Both an Asn-peptide and a Gln-peptide fit well into the hollow active site pocket of yNta1, with the catalytic triad located deeper inside the active site. Specific hydrogen bonds stabilize interactions between N-degron peptides and hydrophobic peripheral regions of the active site pocket. Key determinants for substrate recognition were identified and thereafter confirmed by using structure-based mutagenesis. We also measured affinities between yNta1 (wild-type and its mutants) and specific peptides, and determined KM and kcat for peptides of each type. Together, these results elucidate, in structural and mechanistic detail, specific deamidation mechanisms in the first step of the N-end rule pathway.

The greening after extended darkness1 is an N-end rule pathway mutant with high tolerance to submergence and starvation.

Plants respond to reductions in internal oxygen concentrations with adaptive mechanisms (for example, modifications of metabolism to cope with reduced supply of ATP). These responses are, at the transcriptional level, mediated by the group VII Ethylene Response Factor transcription factors, which have stability that is regulated by the N-end rule pathway of protein degradation. N-end rule pathway mutants are characterized by a constitutive expression of hypoxia response genes and abscisic acid hypersensitivity. Here, we identify a novel proteolysis6 (prt6) mutant allele, named greening after extended darkness1 (ged1), which was previously discovered in a screen for genomes uncoupled-like mutants and shows the ability to withstand long periods of darkness at the seedling stage. Interestingly, this ethyl methanesulfonate-derived mutant shows unusual chromosomal rearrangement instead of a point mutation. Furthermore, the sensitivity of N-end rule pathway mutants ged1 and prt6-1 to submergence was studied in more detail to understand previously contradicting experiments on this topic. Finally, it was shown that mutants for the N-end rule pathway are generally more tolerant to starvation conditions, such as prolonged darkness or submergence, which was partially associated with carbohydrate conservation.

Abstract 94: Nitric Oxide--Regulated RGS4 Loss of Function Coordinates Vascular Growth with Cardiomyocyte Growth in the Absence of External Hypertrophic Stimuli

The mechanisms that coordinate an increase in vasculature with cardiomyocyte growth in adult heart are not known. We report that induction of angiogenesis is sufficient to promote cardiomyocyte growth and myocardial hypertrophy through a novel nitric oxide (NO)-dependent mechanism. Our hypothesis is that an increase in endothelial-derived NO release promotes the proteosomal degradation of negative regulator of G protein signaling RGS4 in cardiomyocytes (CM) thus relieving repression of hypertrophic stimulation through G proteins. Methods include cell culture systems and transgenic mouse models. We generated three conditional cardiac inducible mouse models: (1) to stimulate angiogenesis by transgenic expression of placental growth factor (PlGF); (2) to stimulate angiogenesis by PlGF and compensate for RGS4 degradation by transgenic expression of RGS4 in CM; and (3) to stimulate angiogenesis by PlGF in eNOS -/- mice. Results. In cultured rat neonatal CM, the treatment with a slow release NO donor resulted in increased degradation of RGS4 through the N-end rule pathway of protein degradation. RGS4 loss of function led to increase in cell size through Gbetagamma/PI3Kgamma-dependent Akt/mTORc1 activation. In mice, PlGF expression induced an increase in capillary/myocyte ratio and arteriolar branches (determined by microCT angiograms). Myocardial hypertrophy was induced subsequent to angiogenic stimulation and concurrent with increased in NO production. Consistent with in vitro data hypertrophic stimulation was associated with reduced RGS4 protein levels and robust activation of Akt/mTORc1 signaling. This hypertrophy was prevented by concomitant transgenic expression of RGS4 in CM, despite the persistence of angiogenesis, arteriogenesis, and increased NO release. Furthermore, the NOS inhibitor L-NAME or mTORc1 inhibitor rapamycin significantly attenuated RGS4 degradation, Akt/mTORc1 signaling, and heart size in PlGF-induced mice. A similar effect was observed upon conditional heart-specific PlGF expression in eNOS -/- mice. In conclusion, these findings support a novel NO/RGS4/G protein/Akt mechanism that couples vessel growth with myocyte growth and heart size in the absence of traditional hypertrophic stimuli.

Expression and Biochemical Characterization of the Human Enzyme N-Terminal Asparagine Amidohydrolase

The enzymatic deamidation of N-terminal L-Asn by N-terminal asparagine amidohydrolase (NTAN1) is a feature of the ubiquitin-dependent N-end rule pathway of protein degradation, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. Herein, we report the bacterial expression, purification, and biochemical characterization of human NTAN1 (hNTAN1). We show here that hNTAN1 is highly selective for the hydrolysis of N-terminal peptidyl L-Asn but fails to deamidate free L-Asn or L-Gln, N-terminal peptidyl L-Gln, or acetylated N-terminal peptidyl L-Asn. Similar to other N-terminal deamidases, hNTAN1 is shown to possess a critical Cys residue that is absolutely required for catalysis, corroborated in part by abolishment of activity through the Cys75Ala point mutation. We also present evidence that the exposure of a conserved L-Pro at the N-terminus of hNTAN1 following removal of the initiating L-Met is important for the function of the enzyme. The results presented here should assist in the elucidation of molecular mechanisms underlying the neurological defects of NTAN1-deficient mice observed in other studies, and in the discovery of potential physiological substrates targeted by the enzyme in the modulation of protein turnover via the N-end rule pathway.

Mouse Dfa Is a Repressor of TATA-box Promoters and Interacts with the Abt1 Activator of Basal Transcription

Our study of the mouse Ate1 arginyltransferase, a component of the N-end rule pathway, has shown that Ate1 pre-mRNA is produced from a bidirectional promoter that also expresses, in the opposite direction, a previously uncharacterized gene (Hu, R. G., Brower, C. S., Wang, H., Davydov, I. V., Sheng, J., Zhou, J., Kwon, Y. T., and Varshavsky, A. (2006) J. Biol. Chem. 281, 32559-32573). In this work, we began analyzing this gene, termed Dfa (divergent from Ate1). Mouse Dfa was found to be transcribed from both the bidirectional P(Ate1/Dfa) promoter and other nearby promoters. The resulting transcripts are alternatively spliced, yielding a complex set of Dfa mRNAs that are present largely, although not exclusively, in the testis. A specific Dfa mRNA encodes, via its 3'-terminal exon, a 217-residue protein termed Dfa(A). Other Dfa mRNAs also contain this exon. Dfa(A) is sequelogous (similar in sequence) to a region of the human/mouse HTEX4 protein, whose physiological function is unknown. We produced an affinity-purified antibody to recombinant mouse Dfa(A) that detected a 35-kDa protein in the mouse testis and in several cell lines. Experiments in which RNA interference was used to down-regulate Dfa indicated that the 35-kDa protein was indeed Dfa(A). Furthermore, Dfa(A) was present in the interchromatin granule clusters and was also found to bind to the Ggnbp1 gametogenetin-binding protein-1 and to the Abt1 activator of basal transcription that interacts with the TATA-binding protein. Given these results, RNA interference was used to probe the influence of Dfa levels in luciferase reporter assays. We found that Dfa(A) acts as a repressor of TATA-box transcriptional promoters.

Ablation of arginylation in the mouse N-end rule pathway: loss of fat, higher metabolic rate, damaged spermatogenesis, and neurological perturbations.

In the N-end rule pathway of protein degradation, the destabilizing activity of N-terminal Asp, Glu or (oxidized) Cys residues requires their conjugation to Arg, which is recognized directly by pathway's ubiquitin ligases. N-terminal arginylation is mediated by the Ate1 arginyltransferase, whose physiological substrates include the Rgs4, Rgs5 and Rgs16 regulators of G proteins. Here, we employed the Cre-lox technique to uncover new physiological functions of N-terminal arginylation in adult mice. We show that postnatal deletion of mouse Ate1 (its unconditional deletion is embryonic lethal) causes a rapid decrease of body weight and results in early death of ∼15% of Ate1-deficient mice. Despite being hyperphagic, the surviving Ate1-deficient mice contain little visceral fat. They also exhibit an increased metabolic rate, ectopic induction of the Ucp1 uncoupling protein in white fat, and are resistant to diet-induced obesity. In addition, Ate1-deficient mice have enlarged brains, an enhanced startle response, are strikingly hyperkinetic, and are prone to seizures and kyphosis. Ate1-deficient males are also infertile, owing to defects in Ate1−/− spermatocytes. The remarkably broad range of specific biological processes that are shown here to be perturbed by the loss of N-terminal arginylation will make possible the dissection of regulatory circuits that involve Ate1 and either its known substrates, such as Rgs4, Rgs5 and Rgs16, or those currently unknown.

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

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

Genome-wide screen in yeast to identify modulators of mutant SOD1 toxicity

Yeast (Saccharomyces cerevisiae) cultures can be handled in a partially automated manner, and about 80% of the yeast genes have their homologous counterpart in mammalian cells. Thus, yeast mutant strains are a versatile tool for genetic studies on protein function and toxicity. In order to identify modulators and mechanisms of SOD1 toxicity, we performed a genome wide screen in Saccharomyces cerevisiae. Yeast strains were transformed with wild-type SOD1, as well as several mutant SOD1 species known to cause ALS. A screen for SOD1 toxicity was then performed for each mutant using >4000 haploid mutant yeast strains containing deletions of non-essential genes. We identified 4 yeast homologues of mammalian genes that modified mutant SOD1 toxicity. For example, mutant SOD1-susceptible yeast strains included deletion mutants for a mitochondrial co-chaperone or a key enzyme of the N-end rule pathway of protein degradation. We are currently transferring these findings to higher eukaryotic systems and studying the contribution of the N-end rule pathway-mediated protein degradation for SOD1 toxicity. Moreover, yeast deletion strains susceptible to mutant SOD1 toxicity could potentially be used for automated high-throughput screening of chemical libraries in order to identify therapeutic compounds.

The N-end rule pathway is a sensor of heme

The conjugation of arginine, by arginyl-transferase, to N-terminal aspartate, glutamate or oxidized cysteine is a part of the N-end rule pathway of protein degradation. We report that arginyl-transferase of either the mouse or the yeast Saccharomyces cerevisiae is inhibited by hemin (Fe(3+)-heme). Furthermore, we show that hemin inhibits arginyl-transferase through a redox mechanism that involves the formation of disulfide between the enzyme's Cys-71 and Cys-72 residues. Remarkably, hemin also induces the proteasome-dependent degradation of arginyl-transferase in vivo, thus acting as both a "stoichiometric" and "catalytic" down-regulator of the N-end rule pathway. In addition, hemin was found to interact with the yeast and mouse E3 ubiquitin ligases of the N-end rule pathway. One of substrate-binding sites of the yeast N-end rule's ubiquitin ligase UBR1 targets CUP9, a transcriptional repressor. This site of UBR1 is autoinhibited but can be allosterically activated by peptides that bear destabilizing N-terminal residues and interact with two other substrate-binding sites of UBR1. We show that hemin does not directly occlude the substrate-binding sites of UBR1 but blocks the activation of its CUP9-binding site by dipeptides. The N-end rule pathway, a known sensor of short peptides, nitric oxide, and oxygen, is now a sensor of heme as well. One function of the N-end rule pathway may be to coordinate the activities of small effectors, both reacting to and controlling the redox dynamics of heme, oxygen, nitric oxide, thiols, and other compounds, in part through conditional degradation of specific transcription factors and G protein regulators.

The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators

The conjugation of arginine to proteins is a part of the N-end rule pathway of protein degradation. Three amino (N)-terminal residues--aspartate, glutamate and cysteine--are arginylated by ATE1-encoded arginyl-transferases. Here we report that oxidation of N-terminal cysteine is essential for its arginylation. The in vivo oxidation of N-terminal cysteine, before its arginylation, is shown to require nitric oxide. We reconstituted this process in vitro as well. The levels of regulatory proteins bearing N-terminal cysteine, such as RGS4, RGS5 and RGS16, are greatly increased in mouse ATE1-/- embryos, which lack arginylation. Stabilization of these proteins, the first physiological substrates of mammalian N-end rule pathway, may underlie cardiovascular defects in ATE1-/- embryos. Our findings identify the N-end rule pathway as a new nitric oxide sensor that functions through its ability to destroy specific regulatory proteins bearing N-terminal cysteine, at rates controlled by nitric oxide and apparently by oxygen as well.

PRT1 of Arabidopsis is a ubiquitin protein ligase of the plant N-end rule pathway with specificity for aromatic amino-terminal residues.

The gene PRT1 of Arabidopsis, encoding a 45-kD protein with two RING finger domains, is essential for the degradation of F-dihydrofolate reductase, a model substrate of the N-end rule pathway of protein degradation. We have determined the function of PRT1 by expression in yeast (Saccharomyces cerevisiae). PRT1 can act as a ubiquitin protein ligase in the heterologous host. The identified substrates of PRT1 have an aromatic residue at their amino-terminus, indicating that PRT1 mediates degradation of N-end rule substrates with aromatic termini but not of those with aliphatic or basic amino-termini. Expression of model substrates in mutant and wild-type plants confirmed this substrate specificity. A ligase activity exclusively devoted to aromatic amino-termini of the N-end rule pathway is apparently unique to plants. The results presented also imply that other known substrates of the plant N-end rule pathway are ubiquitylated by one or more different ubiquitin protein ligases.

An Essential Role of N-Terminal Arginylation in Cardiovascular Development

The enzymatic conjugation of arginine to the N-termini of proteins is a part of the ubiquitin-dependent N-end rule pathway of protein degradation. In mammals, three N-terminal residues-aspartate, glutamate, and cysteine-are substrates for arginylation. The mouse ATE1 gene encodes a family of Arg-tRNA-protein transferases (R-transferases) that mediate N-terminal arginylation. We constructed ATE1-lacking mouse strains and found that ATE1-/- embryos die with defects in heart development and in angiogenic remodeling of the early vascular plexus. Through biochemical analyses, we show that N-terminal cysteine, in contrast to N-terminal aspartate and glutamate, is oxidized before its arginylation by R-transferase, suggesting that the arginylation branch of the N-end rule pathway functions as an oxygen sensor.

Domains required for dimerization of yeast Rad6 ubiquitin-conjugating enzyme and Rad18 DNA binding protein.

The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme required for postreplicational repair of UV-damaged DNA and for damage-induced mutagenesis. In addition, Rad6 functions in the N end rule pathway of protein degradation. Rad6 mediates its DNA repair role via its association with Rad18, whose DNA binding activity may target the Rad6-Rad18 complex to damaged sites in DNA. In its role in N end-dependent protein degradation, Rad6 interacts with the UBR1-encoded ubiquitin protein ligase (E3) enzyme. Previous studies have indicated the involvement of N-terminal and C-terminal regions of Rad6 in interactions with Ubr1. Here, we identify the regions of Rad6 and Rad18 that are involved in the dimerization of these two proteins. We show that a region of 40 amino acids towards the C terminus of Rad18 (residues 371 to 410) is sufficient for interaction with Rad6. This region of Rad18 contains a number of nonpolar residues that have been conserved in helix-loop-helix motifs of other proteins. Our studies indicate the requirement for residues 141 to 149 at the C terminus, and suggest the involvement of residues 10 to 22 at the N terminus of Rad6, in the interaction with Rad18. Each of these regions of Rad6 is indicated to form an amphipathic helix.

The N-end rule pathway of protein degradation.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Similar but distinct versions of the N-end rule operate in all organisms examined, from mammals to fungi and bacteria. In eukaryotes, the N-end rule pathway is a part of the ubiquitin system. Ubiquitin is a 76-residue protein whose covalent conjugation to other proteins plays a role in many biological processes, including cell growth and differentiation. I discuss the current understanding of the N-end rule pathway.

The Leucyl/Phenylalanyl-tRNA-Protein Transferase: OVEREXPRESSION AND CHARACTERIZATION OF SUBSTRATE RECOGNITION, DOMAIN STRUCTURE, AND SECONDARY STRUCTURE

Previous work has shown that, in the bacterium Escherichia coli, the aat gene is essential for the degradation of proteins bearing amino-terminal Arg and Lys residues via the N-end rule pathway of protein degradation. We now show that the aat gene encodes directly the leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase). This enzyme catalyzes the transfer of Leu, Phe, and, less efficiently, Met and Trp, from aminoacyl-tRNAs, to the amino terminus of acceptor proteins. We have used the cloned aat gene to overexpress and purify an affinity tagged L/F-transferase. The recombinant L/F-transferase is as active as the previously purified wild type enzyme and contains no detectable RNA component. We have used the recombinant enzyme to demonstrate that both the solubility and substrate specificity, for aminoacyl-tRNA substrates, of the L/F-transferase are dependent on ionic strength conditions and that the modified nucleotides found in natural tRNAs are not essential for recognition by the enzyme. Limited digestion of the L/F-transferase with trypsin removes the proline rich NH2 terminus of the enzyme identifying a globular core, and circular dichroism demonstrates that the L/F-transferase is predominantly alpha-helical. Finally, a region of sequence conservation between the L/F-transferase and the NH2-terminal protein acetylases has been identified.

The proteasome/multicatalytic—multifunctional proteinase In vivo function in the ubiquitin-dependent N-end rule pathway of protein degradation in eukaryotes

Proteinase yscE, the proteasome/multicatalytic—multifunctional proteinase of yeast had been shown to function in stress response and in the degradation of ubiquitinated proteins [(1991) EMBO J. 10, 555–562]. A well-defined set of proteins degraded via ubiquitin-mediated proteolysis are the substrates of the N-end rule pathway [(1986) Science 234, 179–186; (1989) Science 243, 1576–1583]. We show that mutants defective in the chymotryptic activity of proteinase yscE fail to degrade substrates of the N-end rule pathway. This gives further proof of the proteasome being a central catalyst in ubiquitin-mediated proteolysis.

Cloning and functional analysis of the arginyl-tRNA-protein transferase gene ATE1 of Saccharomyces cerevisiae.

Aminoacyl-tRNA-protein transferases (Arg-transferases) catalyze post-translational conjugation of specific amino acids to the amino termini of acceptor proteins. A function of these enzymes in eukaryotes has been shown to involve the conjugation of destabilizing amino acids to the amino termini of short-lived proteins, these reactions being a part of the N-end rule pathway of protein degradation (Gonda, D. K., Bachmair, A., Wünning, I., Tobias, J. W., Lane, W. S., and Varshavsky, A. (1989) J. Biol. Chem. 264, 16700-16712). We have cloned the ATE1 gene of the yeast Saccharomyces cerevisiae which encodes arginyl-tRNA-protein transferase. ATE1 gives rise to a approximately 1.6-kilobase mRNA and codes for a 503-residue protein. Expression of the yeast ATE1 gene in Escherichia coli, which lacks Arg-transferases, was used to show that the ATE1 protein possesses the Arg-transferase activity. Null ate1 mutants are viable but lack the Arg-transferase activity and are unable to degrade those substrates of the N-end rule pathway that start with residues recognized by the Arg-transferase.