Marker for real-time analysis of caspase activity in intact cells

Apoptosis, or programmed cell death, is an important regulator of growth, development, defense, and homeostasis in multicellular organisms. A family of cysteine proteases known as caspases is central to many apoptotic pathways, and thus detection of their activity offers an effective means to assess apoptosis. However, currently available methods only allow the evaluation of in vivo caspase activity at a given time point or over a few hours. To assess the activity over extended periods of time, we designed a novel, real-time, in vivo marker that utilizes the N-end rule degradation pathway to allow the detection of caspase activity as reflected by increasing enhanced GFP (EGFP) stability. The marker has an N-terminal arginine in the absence of caspase activity and is rapidly degraded. In vivo caspase activity removes the marker's N-terminal arginine residue, leaving an EGFP with an N-terminal methionine that results in stable fluorescence. In our study, the marker accurately depicted an increase in caspase activity in apoptotic cells and also detected significant endogenous caspase activity in non-apoptotic cells. The downstream effects of this endogenous activity detected in intact, nonapoptotic cells must be regulated by the cell preventing apoptosis. These studies also demonstrate the feasibility of using the N-end rule to study endogenous enzymatic activities other than those associated with proteasomal degradation.     

Crystal Structure of Human Protein N-Terminal Glutamine Amidohydrolase,an Initial Component of the N-End Rule Pathway

The N-end rule states that half-life of protein is determined by their N-terminal amino acid residue. N-terminal glutamine amidohydrolase (Ntaq) converts N-terminal glutamine to glutamate by eliminating the amine group and plays an essential role in the N-end rule pathway for protein degradation. Here, we report the crystal structure of human Ntaq1 bound with the N-terminus of a symmetry-related Ntaq1 molecule at 1.5 Å resolution. The structure reveals a monomeric globular protein with alpha-beta-alpha three-layer sandwich architecture. The catalytic triad located in the active site, Cys-His-Asp, is highly conserved among Ntaq family and transglutaminases from diverse organisms. The N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft and the active site suggest possible substrate binding mode of hNtaq1. Based on our crystal structure of hNtaq1 and docking study with all the tripeptides with N-terminal glutamine, we propose how the peptide backbone recognition patch of hNtaq1 forms nonspecific interactions with N-terminal peptides of substrate proteins. Upon binding of a substrate with N-terminal glutamine, active site catalytic triad mediates the deamination of the N-terminal residue to glutamate by a mechanism analogous to that of cysteine proteases.     

Dissection of c-MOS degron

c-MOS, a MAP kinase kinase kinase, is a regulator of oocyte maturation. The concentration of c-MOS is controlled in part through its conditional degradation. Previous studies proposed the "second-codon rule", according to which the N-terminal proline (Pro) of c-MOS is a destabilizing residue that targets c-MOS for degradation. We analyzed the degradation signal (degron) of c-MOS in Xenopus oocytes, found it to be a portable degron, and demonstrated that, contrary to the model above, the N-terminal Pro residue of c-MOS is entirely dispensable for its degradation if Ser-2 (encoded Ser-3) of c-MOS is replaced by a small non-phosphorylatable residue such as Gly. The dependence of c-MOS degradation on N-terminal Pro is shown to be caused by a Pro-mediated downregulation of the net phosphorylation of Ser-2, a modification that halts c-MOS degradation in oocytes. Thus, the N-terminal Pro residue of c-MOS is not a recognition determinant for a ubiquitin ligase, in agreement with earlier evidence that Pro is a stabilizing residue in the N-end rule.     

Altered activity, social behavior, and spatial memory in mice lacking the NTAN1p amidase and the asparagine branch of the N-end rule pathway

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. N-terminal asparagine and glutamine are tertiary destabilizing residues, in that they are enzymatically deamidated to yield secondary destabilizing residues aspartate and glutamate, which are conjugated to arginine, a primary destabilizing residue. N-terminal arginine of a substrate protein is bound by the Ubr1-encoded E3alpha, the E3 component of the ubiquitin-proteasome-dependent N-end rule pathway. We describe the construction and analysis of mouse strains lacking the asparagine-specific N-terminal amidase (Nt(N)-amidase), encoded by the Ntan1 gene. In wild-type embryos, Ntan1 was strongly expressed in the branchial arches and in the tail and limb buds. The Ntan1(-/-) mouse strains lacked the Nt(N)-amidase activity but retained glutamine-specific Nt(Q)-amidase, indicating that the two enzymes are encoded by different genes. Among the normally short-lived N-end rule substrates, only those bearing N-terminal asparagine became long-lived in Ntan1(-/-) fibroblasts. The Ntan1(-/-) mice were fertile and outwardly normal but differed from their congenic wild-type counterparts in spontaneous activity, spatial memory, and a socially conditioned exploratory phenotype that has not been previously described with other mouse strains.     

ATP-driven self-assembly of a morphogenetic protein in Bacillus subtilis

The N-end rule targets specific proteins for destruction in prokaryotes and eukaryotes. Here, we report a crystal structure of a bacterial N-end rule adaptor, ClpS, bound to a peptide mimic of an N-end rule substrate. This structure, which was solved at a resolution of 1.15 A, reveals specific recognition of the peptide alpha-amino group via hydrogen bonding and shows that the peptide's N-terminal tyrosine side chain is buried in a deep hydrophobic cleft that pre-exists on the surface of ClpS. The adaptor side chains that contact the peptide's N-terminal residue are highly conserved in orthologs and in E3 ubiquitin ligases that mediate eukaryotic N-end rule recognition. We show that mutation of critical ClpS contact residues abrogates substrate delivery to and degradation by the AAA+ protease ClpAP, demonstrate that modification of the hydrophobic pocket results in altered N-end rule specificity, and discuss functional implications for the mechanism of substrate delivery.     

Degradation of HIV-1 integrase by the N-end rule pathway

Human immunodeficiency virus type-1 (HIV-1) integrase catalyzes the irreversible insertion of the viral genome into host chromosomal DNA. We have developed a mammalian expression system for the synthesis of authentic HIV-1 integrase in the absence of other viral proteins. Integrase, which bears a N-terminal phenylalanine, was found to be a short-lived protein in human embryo kidney 293T cells. The degradation of integrase could be suppressed by proteasome inhibitors. N-terminal phenylalanine is recognized as a degradation signal by a ubiquitin-proteasome proteolytic system known as the N-end rule pathway. The replacement of N-terminal phenylalanine with methionine, valine, or glycine, which are stabilizing residues in the N-end rule, resulted in metabolically stabilized integrase proteins (half-life of N-terminal Met-integrase was at least 3 h). Conversely, the substitution of N-terminal phenylalanine with other destabilizing residues retained the metabolic instability of integrase. These findings indicate that the HIV-1 integrase is a physiological substrate of the N-end rule. We discuss a possible functional similarity to the better understood turnover of the bacteriophage Mu transposase and functions of integrase instability to the maintenance and integrity of the host cell genome.     

The N-end rule in Escherichia coli: cloning and analysis of the leucyl, phenylalanyl-tRNA-protein transferase gene aat.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Distinct versions of the N-end rule operate in bacteria, fungi, and mammals. We report the cloning and analysis of aat, the Escherichia coli gene that encodes leucyl, phenylalanyl-tRNA-protein transferase (L/F-transferase), a component of the bacterial N-end rule pathway. L/F-transferase is required for the degradation of N-end rule substrates bearing an N-terminal arginine or lysine. The aat gene maps to the 19-min region of the E. coli chromosome and encodes a 234-residue protein whose sequence lacks significant similarities to sequences in data bases. In vitro, L/F-transferase catalyzes the posttranslational conjugation of leucine or phenylalanine to the N termini of proteins that bear an N-terminal arginine or lysine. However, the isolation and sequence analysis of a beta-galactosidase variant engineered to expose an N-terminal arginine in vivo revealed the conjugation of leucine but not of phenylalanine to the N terminus of the beta-galactosidase variant. Thus, the specificity of L/F-transferase in vivo may be greater than that in vitro. The aat gene is located approximately 1 kb from clpA, which encodes a subunit of ATP-dependent protease Clp. Although both aat and clpA are required for the degradation of certain N-end rule substrates, their nearly adjacent genes are convergently transcribed. The aat gene lies downstream of an open reading frame that encodes a homolog of the mammalian multidrug resistance P glycoproteins.     

Tuning the strength of a bacterial N-end rule degradation signal

The N-end rule is a degradation pathway conserved from bacteria to mammals that links a protein's stability in vivo to the identity of its N-terminal residue. In Escherichia coli, the components of this pathway directly responsible for protein degradation are the ClpAP protease and its adaptor ClpS. We recently demonstrated that ClpAP is able to recognize N-end motifs in the absence of ClpS although with significantly reduced substrate affinity. In this study, a systematic sequence analysis reveals new features of N-end rule degradation signals. To achieve specificity, recognition of an N-end motif by the protease-adaptor complex uses both the identity of the N-terminal residue and a free alpha-amino group. Acidic residues near the first residue decrease substrate affinity, demonstrating that the identity of adjacent residues can affect recognition although significant flexibility is tolerated. However, shortening the distance between the N-end residue and the stably folded portion of a protein prevents degradation entirely, indicating that an N-end signal alone is not always sufficient for degradation. Together, these data define in vitro the sequence and structural requirements for the function of bacterial N-end signals.     

ClpS is the recognition component for Escherichia coli substrates of the N-end rule degradation pathway

The N-end rule degradation pathway states that the half-life of a protein is determined by the nature of its N-terminal residue. In Escherichia coli the adaptor protein ClpS directly interacts with destabilizing N-terminal residues and transfers them to the ClpA/ClpP proteolytic complex for degradation. The crucial role of ClpS in N-end rule degradation is currently under debate, since ClpA/ClpP was shown to process selected N-terminal degrons harbouring destabilizing residues in the absence of ClpS. Here, we investigated the contribution of ClpS to N-end rule degradation by two approaches. First, we performed a systematic mutagenesis of selected N-degron model substrates, demonstrating that ClpS but not ClpA specifically senses the nature of N-terminal residues. Second, we identified two natural N-end rule substrates of E. coli : Dps and PATase (YgjG). The in vivo degradation of both proteins strictly relied on ClpS, thereby establishing the function of ClpS as the essential discriminator of the E. coli N-end rule pathway.     

The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases

Substrates of the N-end rule pathway are recognized by the Ubr1 E3 ubiquitin ligase through their destabilizing amino-terminal residues. Our previous work showed that the Ubr1 E3 and the Ufd4 E3 together target an internal degradation signal (degron) of the Mgt1 DNA repair protein. Ufd4 is an E3 enzyme of the ubiquitin-fusion degradation (UFD) pathway that recognizes an N-terminal ubiquitin moiety. Here we show that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. Although Ubr1 can recognize and polyubiquitylate an N-end rule substrate in the absence of Ufd4, the Ubr1-Ufd4 complex is more processive in that it produces a longer substrate-linked polyubiquitin chain. Conversely, Ubr1 can function as a polyubiquitylation-enhancing component of the Ubr1-Ufd4 complex in its targeting of UFD substrates. We also found that Ubr1 can recognize the N-terminal ubiquitin moiety. These and related advances unify two proteolytic systems that have been studied separately for two decades.     

The N-end rule pathway: emerging functions and molecular principles 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 C-terminal proteolytic fragment of the breast cancer susceptibility type 1 protein (BRCA1) is degraded by the N-end rule pathway

The breast cancer susceptibility type 1 gene product (BRCA1) is cleaved by caspases upon the activation of apoptotic pathways. After proteolysis the C-terminal fragment has been reported to translocate to the cytoplasm and promote cell death. Here we report that the C-terminal fragment is unstable in cells as it is targeted for degradation by the N-end rule pathway. The data reveals that mutating the wild type N-terminal aspartate, of the C-terminal fragment, to valine stabilizes the fragment. If the N terminus is mutated to another N-terminal destabilizing residue, like arginine, the C-terminal fragment remains unstable in cells. Last, the C-terminal fragment of BRCA1 is stable in cells lacking ATE1, a component of the N-end rule pathway.     

Killing of macrophages by anthrax lethal toxin: involvement of the N-end rule pathway

Macrophages from certain inbred mouse strains are rapidly killed (< 90 min) by anthrax lethal toxin (LT). LT cleaves cytoplasmic MEK proteins at 20 min and induces caspase-1 activation in sensitive macrophages at 50-60 min, but the mechanism of LT-induced death is unknown. Proteasome inhibitors block LT-mediated caspase-1 activation and can protect against cell death, indicating that the degradation of at least one cellular protein is required for LT-mediated cell death. Proteins can be degraded by the proteasome via the N-end rule, in which a protein's stability is determined by its N-terminal residue. Using amino acid derivatives that act as inhibitors of this pathway, we show that the N-end rule is required for LT-mediated caspase-1 activation and cell death. We also found that bestatin methyl ester, an aminopeptidase inhibitor protects against LT in vitro and in vivo and that the different inhibitors of the protein degradation pathway act synergistically in protecting against LT. We identify c-IAP1, a mammalian member of the inhibitor of apoptosis protein (IAP) family, as a novel N-end rule substrate degraded in macrophages treated with LT. We also show that LT-induced c-IAP1 degradation is independent of the IAP-antagonizing proteins Smac/DIABLO and Omi/HtrA2, but dependent on caspases.     

The N-end rule pathway for regulated proteolysis: prokaryotic and eukaryotic strategies

The N-end rule states that the half-life of a protein is determined by the nature of its N-terminal residue. This fundamental principle of regulated proteolysis is conserved from bacteria to mammals. Although prokaryotes and eukaryotes employ distinct proteolytic machineries for degradation of N-end rule substrates, recent findings indicate that they share common principles of substrate recognition. In eukaryotes substrate recognition is mediated by N-recognins, a class of E3 ligases that labels N-end rule substrates via covalent linkage to ubiquitin, allowing the subsequent substrate delivery to the 26S proteasome. In bacteria, the adaptor protein ClpS exhibits homology to the substrate binding site of N-recognin. ClpS binds to the destabilizing N-termini of N-end rule substrates and directly transfers them to the ClpAP protease.     

The ubiquitin ligase Ubr2, a recognition E3 component of the N-end rule pathway, stabilizes Tex19.1 during spermatogenesis

Ubiquitin E3 ligases target their substrates for ubiquitination, leading to proteasome-mediated degradation or altered biochemical properties. The ubiquitin ligase Ubr2, a recognition E3 component of the N-end rule proteolytic pathway, recognizes proteins with N-terminal destabilizing residues and plays an important role in spermatogenesis. Tex19.1 (also known as Tex19) has been previously identified as a germ cell-specific protein in mouse testis. Here we report that Tex19.1 forms a stable protein complex with Ubr2 in mouse testes. The binding of Tex19.1 to Ubr2 is independent of the second position cysteine of Tex19.1, a putative target for arginylation by the N-end rule pathway R-transferase. The Tex19.1-null mouse mutant phenocopies the Ubr2-deficient mutant in three aspects: heterogeneity of spermatogenic defects, meiotic chromosomal asynapsis, and embryonic lethality preferentially affecting females. In Ubr2-deficient germ cells, Tex19.1 is transcribed, but Tex19.1 protein is absent. Our results suggest that the binding of Ubr2 to Tex19.1 metabolically stabilizes Tex19.1 during spermatogenesis, revealing a new function for Ubr2 outside the conventional N-end rule pathway.     

Construction and Analysis of Mouse Strains Lacking the Ubiquitin Ligase UBR1 (E3α) of the N-End Rule Pathway

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, the UBR1-encoded ubiquitin ligase (E3) of the N-end rule pathway mediates the targeting of substrate proteins in part through binding to their destabilizing N-terminal residues. The functions of the yeast N-end rule pathway include fidelity of chromosome segregation and the regulation of peptide import. Our previous work described the cloning of cDNA and a gene encoding the 200-kDa mouse UBR1 (E3alpha). Here we show that mouse UBR1, in the presence of a cognate mouse ubiquitin-conjugating (E2) enzyme, can rescue the N-end rule pathway in ubr1Delta S. cerevisiae. We also constructed UBR1(-/-) mouse strains that lacked the UBR1 protein. UBR1(-/-) mice were viable and fertile but weighed significantly less than congenic +/+ mice. The decreased mass of UBR1(-/-) mice stemmed at least in part from smaller amounts of the skeletal muscle and adipose tissues. The skeletal muscle of UBR1(-/-) mice apparently lacked the N-end rule pathway and exhibited abnormal regulation of fatty acid synthase upon starvation. By contrast, and despite the absence of the UBR1 protein, UBR1(-/-) fibroblasts contained the N-end rule pathway. Thus, UBR1(-/-) mice are mosaics in regard to the activity of this pathway, owing to differential expression of proteins that can substitute for the ubiquitin ligase UBR1 (E3alpha). We consider these UBR1-like proteins and discuss the functions of the mammalian N-end rule pathway.     

The ClpS adaptor mediates staged delivery of N-end rule substrates to the AAA+ ClpAP protease

The ClpS adaptor delivers N-end rule substrates to ClpAP, an energy-dependent AAA+ protease, for degradation. How ClpS binds specific N-end residues is known in atomic detail and clarified here, but the delivery mechanism is poorly understood. We show that substrate binding is enhanced when ClpS binds hexameric ClpA. Reciprocally, N-end rule substrates increase ClpS affinity for ClpA(6). Enhanced binding requires the N-end residue and a peptide bond of the substrate, as well as multiple aspects of ClpS, including a side chain that contacts the substrate α-amino group and the flexible N-terminal extension (NTE). Finally, enhancement also needs the N domain and AAA+ rings of ClpA, connected by a long linker. The NTE can be engaged by the ClpA translocation pore, but ClpS resists unfolding/degradation. We propose a staged-delivery model that illustrates how intimate contacts between the substrate, adaptor, and protease reprogram specificity and coordinate handoff from the adaptor to the protease.     

Stability of the Listeria monocytogenes ActA protein in mammalian cells is regulated by the N-end rule pathway

Upon infection of mammalian cells, Listeria monocytogenes lyses the phagosome and enters the cytosol, where it secretes proteins necessary for its intracellular growth cycle. Consequently, bacterial proteins exposed to the cytosol are potential targets for degradation by host cytosolic proteases. One pathway for degradation of host cytosolic proteins, the N-end rule pathway, involves recognition of the N-terminal amino acid and is mediated by the proteasome. However, very few natural N-end rule substrates have been identified. We have examined the L. monocytogenes ActA protein as a potential target for this pathway. ActA is an essential determinant of L. monocytogenes pathogenesis that is required to induce actin-based motility and cell-to-cell spread. We show that the half-life of a secreted form of ActA can be altered in the mammalian cytosol by changing the N-terminal amino acid. Moreover, the introduction of a destabilizing N-terminus into the functional, surface-bound form of ActA results in a small-plaque phenotype in L2 cells, which is partially reversible by an inhibitor of the proteasome. These results indicate that the L. monocytogenes ActA protein is a natural N-end rule substrate, and that optimal function of ActA in mediating cell-to-cell spread is dependent upon its intracellular turnover rate.     

Degradation of ornithine decarboxylase in reticulocyte lysate is ATP-dependent but ubiquitin-independent

Reticulocyte lysate contains all the components of the ubiquitin-dependent proteolytic system. Several proteins are degraded in reticulocyte lysate in a ubiquitin-dependent manner. However, none of the proteins studied has a short intracellular half-life. We have investigated the degradation of ornithine decarboxylase (ODC), one of the most labile proteins in mammalian cells. ODC is efficiently degraded in reticulocyte lysate depleted of the ubiquitin activating enzyme, E1, in fraction II of reticulocyte lysate completely lacking ubiquitin, and in fraction II depleted of the entire complex of enzymes responsible for the ligation of ubiquitin to target proteins. The degradation of ODC is ATP dependent. Therefore, our results demonstrate that in addition to the ubiquitin-dependent proteolytic pathway, reticulocyte lysate contains at least one additional ATP-dependent proteolytic pathway. In vitro synthesized ODC served as a substrate in the present degradation study. Its successful utilization establishes a general strategy for investigating the degradation of short-lived proteins (for which a corresponding cDNA is available), that constitute a very small fraction of cellular proteins and for which purification is difficult or impossible. In contrast to ODC synthesized in vitro, that isolated from cells was not degraded by the reticulocyte lysate degradation system, suggesting that post-translational modifications may be involved in regulating ODC degradation.     

The effect of canavanine on protein synthesis and protein degradation in IMR-90 fibroblasts

Proteins of IMR-90 fibroblasts incorporating [³⁵S]methionine during a 1 h labelling period in the presence of the arginine analogue canavanine were degraded twice as rapidly in the cells as were proteins similarly made in the presence of arginine. Using both isoelectric focusing and SDS-polyacrylamide gel electrophoretic analyses, the banding patterns of proteins labelled in the presence of canavanine and arginine were found to differ. This banding difference was detected as early as 15 min after canavanine treatment. With the exception of one minor band in isoelectric focusing gel, the relative intensity of labelled protein bands for the control samples remained unchanged during the 2 h period of protein degradation being investigated. This was also true for the proteins labelled in the presence of canavanine, despite the increase in their rate of degradation. Banding difference between canavanine and arginine treatment was also detected in an in vitro reticulocyte lysate translation system dependent on fibroblast mRNA. Proteins labelled in the presence of a different analogue, p-fluorophenylalanine instead of phenylalanine, however, had similar banding patterns as the control both in the lysate system and in intact cells.