Protein N-terminal Acetyltransferases Act as N-terminal Propionyltransferases In Vitro and In Vivo

N-terminal acetylation (Nt-acetylation) is a highly abundant protein modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs), which transfer an acetyl group from acetyl coenzyme A to the alpha amino group of a nascent polypeptide. Nt-acetylation has emerged as an important protein modifier, steering protein degradation, protein complex formation and protein localization. Very recently, it was reported that some human proteins could carry a propionyl group at their N-terminus. Here, we investigated the generality of N-terminal propionylation by analyzing its proteome-wide occurrence in yeast and we identified 10 unique in vivo Nt-propionylated N-termini. Furthermore, by performing differential N-terminome analysis of a control yeast strain (yNatA), a yeast NatA deletion strain (yNatAΔ) or a yeast NatA deletion strain expressing human NatA (hNatA), we were able to demonstrate that in vivo Nt-propionylation of several proteins, displaying a NatA type substrate specificity profile, depended on the presence of either yeast or human NatA. Furthermore, in vitro Nt-propionylation assays using synthetic peptides, propionyl coenzyme A, and either purified human NATs or immunoprecipitated human NatA, clearly demonstrated that NATs are Nt-propionyltransferases (NPTs) per se. We here demonstrate for the first time that Nt-propionylation can occur in yeast and thus is an evolutionarily conserved process, and that the NATs are multifunctional enzymes acting as NPTs in vivo and in vitro, in addition to their main role as NATs, and their potential function as lysine acetyltransferases (KATs) and noncatalytic regulators.Modifications greatly increases a cell''s proteome diversity confined by the natural amino acids. As more than 80% of human proteins, more than 70% of plant and fly proteins and more than 60% of yeast proteins are N-terminally acetylated (Nt-acetylated),¹ this modification represents one of the most common protein modifications in eukaryotes (1–5). Recent studies have pointed to distinct functional consequences of Nt-acetylation (6): creating degradation signals recognized by a ubiquitin ligase of a new branch of the N-end rule pathway (7), preventing translocation across the endoplasmic reticulum membrane (8), and mediating protein complex formation (9). Nt-acetylation further appears to be essential for life in higher eukaryotes; for instance, a mutation in the major human N-terminal acetyltransferase (NAT), hNatA, was recently shown to be the cause of Ogden syndrome by which male infants are underdeveloped and die at infancy (10). Unlike lysine acetylation, Nt-acetylation is considered an irreversible process, and further, to mainly occur on the ribosome during protein synthesis (11–15). In yeast and humans, three NAT complexes are responsible for the majority of Nt-acetylation; NatA, NatB and NatC, each of which has a defined substrate specificity (16). NatA acetylates Ser-, Ala-, Gly-, Thr-, Val- and Cys- N-termini generated on removal of the initiator methionine (iMet) (1, 17–19). NatB and NatC acetylate N-termini in which the iMet is followed by an acidic (20–23) or a hydrophobic residue respectively (24–26). Naa40p/NatD was shown to acetylate the Ser-starting N-termini of histones H2A and H4 (27, 28). NatE, composed of the catalytic Naa50p (Nat5p) has substrate specificity toward iMet succeeded by a hydrophobic amino acid (29, 30). As largely the same Nt-acetylation patterns are found in yeast and humans, it was believed that the NAT-machineries were conserved in general (31). However, the recently discovered higher eukaryotic specific NAT, Naa60p/NatF, was found to display a partially distinct substrate specificity in part explaining the higher degree of Nt-acetylation in higher versus lower eukaryotes (4).Human NatA is composed of two main subunits: the catalytic subunit hNaa10p and the auxiliary subunit, hNaa15p that is presumably responsible for anchoring the complex to the ribosome (14, 19). The chaperone-like HYPK protein is also stably associated with the NatA subunits and may be essential for efficient NatA activity (32). In addition, hNaa50p was shown to be physically associated with hNatA, however it is believed not to affect NatA activity (14, 33, 34). hNaa50p was also shown to exhibit N^ε-acetyltransferase (KAT) activity (29), however, the structure of hNaa50p with its peptide substrate bound strongly indicates that the peptide binding pocket is specifically suited to accommodate N-terminal peptides, as opposed to lysine residues (35). The human NatA subunits are associated with ribosomes, but interestingly, significant fractions are also nonribosomal (19, 30, 32). Of further notice, the catalytic subunits, hNaa10p and hNaa50p, were also found to partially act independently of the hNatA complex (30, 36).Recent studies have identified novel in vivo acyl modifications of proteins. Mass spectrometry data of affinity-enriched acetyllysine-containing peptides from HeLa cells showed the presence of propionylated and butyrylated lysines in histone H4 peptides (37). Similar analyses also showed the presence of propionylated lysines in p53, p300 and CREB-binding protein (38) besides the yeast histones H2B, H3 and H4 (39). Propionylated or butyrylated residues differ by only one or two extra methyl moieties as compared with their acetylated counterparts, thereby adding more hydrophobicity and bulkiness to the affected residue. To date, no distinct propionyl- or butyryltransferases responsible for these modifications have been identified. However, by using propionyl coenzyme A (Prop-CoA) or butyryl coenzyme A (But-CoA) as donors in the enzyme reaction, it was shown that some of the previously characterized lysine acetyltransferases (KATs) are able to respectively catalyze propionylation and butyrylation of lysine residues both in vitro (37, 40–42) and in vivo (38, 41). Similarly, it has been shown that lysine deacetylases also are capable of catalyzing depropionylation (40, 41, 43, 44) and debutyrylation (44) (see review (45)).Interestingly, mass spectrometry data also suggested that propionylated N-termini are present in human cell lines (46, 47). Until today, an N-terminal propionyl transferase (NPT) catalyzing N-terminal propionylation (Nt-propionylation) has to our knowledge not been identified.In this study, we hypothesized that NATs might have the ability to act as NPTs. In vitro experiments using purified hNaa10p, hNaa50p or immunoprecipitated human NatA complex indeed confirmed their intrinsic capacity to catalyze Nt-propionylation toward synthetic peptides. NatA was also found capable of Nt-butyrylation in vitro. By means of N-terminomics, we further investigated the presence of yeast Nt-propionylated proteins in vivo. Indeed, we found evidence for Nt-propionylation being a naturally occurring modification in yeast. Interestingly, in a yeast strain lacking NatA, we observed a loss in Nt-propionylation and Nt-acetylation for several NatA substrates, as compared with a control yeast strain expressing endogenous NatA or a strain ectopically expressing hNatA. Thus, besides acting as NATs, yeast and human NatA can act as NPTs and we thus demonstrate for the first time that NATs have the capacity of both acetylating and propionylating protein N-termini in vivo and in vitro.     

In Vitro and In Vivo Biology of Recombinant Adenovirus Vectors with E1, E1/E2A,or E1/E4 Deleted

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.Replication-deficient human adenoviruses (Ad) have been widely investigated as ex vivo and in vivo gene delivery systems for human gene therapy. The ability of these vectors to mediate the efficient expression of candidate therapeutic or vaccine genes in a variety of cell types, including postmitotic cells, is considered an advantage over other gene transfer vectors (3, 28, 49). However, the successful application of currently available E1-defective Ad vectors in human gene therapy has been hampered by the fact that transgene expression is only transient in vivo (2, 15, 16, 33, 36, 46). This short-lived in vivo expression of the transgene has been explained, at least in part, by the induction in vivo of cytotoxic immune responses to cells infected with the Ad vector. Studies with rodent systems have suggested that cytotoxic T lymphocytes (CTLs) directed against virus antigens synthesized de novo in the transduced tissues play a major role in eliminating cells containing the E1-deleted viral genome (56–58, 61). Consistent with the concept of cellular antiviral immunity, expression of transgenes is significantly extended in experimental rodent systems that are deficient in various components of the cellular immune system or that have been rendered immunocompromised by administration of pharmacological agents (2, 33, 37, 48, 60, 64).Based on the assumption that further reduction of viral antigen expression may lower the immune response and thus extend persistence of transgene expression, previous studies have investigated the consequences of deleting both E1 and an additional viral regulatory region, such as E2A or E4. The E2A region encodes a DNA binding protein (DBP) with specific affinity for single-stranded Ad DNA. The DNA binding function is essential for the initiation and elongation of viral DNA synthesis during the early phase of Ad infection. During the late phase of infection, DBP plays a central role in the activation of the major late promoter (MLP) (for a recent review, see reference 44). The E4 region, located at the right end of the viral genome, encodes several regulatory proteins with pleiotropic functions which are involved in the accumulation, splicing, and transport of early and late viral mRNAs, in DNA replication, and in virus particle assembly (reviewed in reference 44). The simultaneous deletion of E1 and E2A or of E1 and E4 should therefore further reduce the replication of the virus genome and the expression of early and late viral genes. Such multidefective vectors have been generated and tested in vitro and in vivo (9, 12, 17, 19–21, 23, 24, 26, 34, 40, 52, 53, 59, 62, 63). Recombinant vectors with E1 deleted and carrying an E2A temperature-sensitive mutation (E2Ats) have been shown in vitro to express much smaller amounts of virus proteins, leading to extended transgene expression in cotton rats and mice (19, 20, 24, 59). To eliminate the risks of reversion of the E2Ats point mutation to a wild-type phenotype, improved vectors with both E1 and E2A deleted were subsequently generated in complementation cell lines coexpressing E1 and E2A genes (26, 40, 63). In vitro analysis of human cells infected by these viruses demonstrated that the double deletion completely abolished viral DNA replication and late protein synthesis (26). Similarly, E1/E4-deleted vectors have been generated in various in vitro complementation systems and tested in vitro and in vivo (9, 17, 23, 45, 52, 53, 62). These studies showed that deletion of both E1 and E4 did indeed reduce significantly the expression of early and late virus proteins (17, 23), leading to a decreased anti-Ad host immune response (23), reduced hepatotoxicity (17, 23, 52), and improved in vivo persistence of the transduced liver cells (17, 23, 52).Interpretation of these results is difficult, however, since all tested E1- and E1/E4-deleted vectors encoded the bacterial β-galactosidase (βgal) marker, whose strong immunogenicity is known to influence the in vivo persistence of Ad-transduced cells (32, 37). Moreover, the results described above are not consistent with the conclusions from other studies showing, in various immunocompetent mouse models, that cellular immunity to Ad antigens has no detectable impact on the persistence of the transduced cells (37, 40, 50, 51). Furthermore, in contrast to results of earlier studies (19, 20, 59), Fang et al. (21) demonstrated that injection of E1-deleted/E2Ats vectors into immunocompetent mice and hemophilia B dogs did not lead to an improvement of the persistence of transgene expression compared to that with isogenic E1-deleted vectors. Similarly, Morral et al. (40) did not observe any difference in persistence of transgene expression in mice injected with either vectors deleted in E1 only or vectors deleted in both E1 and E2A. Finally, the demonstration that some E4-encoded products can modulate transgene expression (1, 17, 36a) makes the evaluation of E1- and E1/E4-deleted vectors even more complex when persistence of transgene expression is used for direct comparison of the in vivo persistence of cells transduced by the two types of vectors.The precise influence of the host immune response to viral antigens on the in vivo persistence of the transduced cells, and hence the impact of further deletions in the virus genome, therefore still remains unclear. To investigate these questions, we generated a set of isogenic vectors with single deletions (AdE1°) and double deletions (AdE1°E2A° and AdE1°E4°) and their corresponding complementation cell lines and compared the biologies and immunogenicities of these vectors in vitro and in vivo. To eliminate any possible influence of transgene-encoded products on the interpretation of the in vivo results, we used E1-, E1/E2A-, and E1/E4-deleted vectors with no transgenes.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]