Arginyltransferase (Ate1) regulates the RGS7 protein level and the sensitivity of light-evoked ON-bipolar responses.

Regulator of G-protein signaling 7 (RGS7) is predominately present in the nervous system and is essential for neuronal signaling involving G-proteins. Prior studies in cultured cells showed that RGS7 is regulated via proteasomal degradation, however no protein is known to facilitate proteasomal degradation of RGS7 and it has not been shown whether this regulation affects G-protein signaling in neurons. Here we used a knockout mouse model with conditional deletion of arginyltransferase (Ate1) in the nervous system and found that in retinal ON bipolar cells, where RGS7 modulates a G-protein to signal light increments, deletion of Ate1 raised the level of RGS7. Electroretinographs revealed that lack of Ate1 leads to increased light-evoked response sensitivities of ON-bipolar cells, as well as their downstream neurons. In cultured mouse embryonic fibroblasts (MEF), RGS7 was rapidly degraded via proteasome pathway and this degradation was abolished in Ate1 knockout MEF. Our results indicate that Ate1 regulates RGS7 protein level by facilitating proteasomal degradation of RGS7 and thus affects G-protein signaling in neurons.

ATE1-Mediated Post-Translational Arginylation Is an Essential Regulator of Eukaryotic Cellular Homeostasis.

Arginylation is a protein post-translational modification catalyzed by arginyl-tRNA transferases (ATE1s), which are critical enzymes conserved across all eukaryotes. Arginylation is a key step in the Arg N-degron pathway, a hierarchical cellular signaling pathway that links the ubiquitin-dependent degradation of a protein to the identity of its N-terminal amino acid side chain. The fidelity of ATE1-catalyzed arginylation is imperative, as this post-translational modification regulates several essential biological processes such as cardiovascular maturation, chromosomal segregation, and even the stress response. While the process of ATE1-catalyzed arginylation has been studied in detail at the cellular level, much remains unknown about the structure of this important enzyme, its mechanism of action, and its regulation. In this work, we detail the current state of knowledge on ATE1-catalyzed arginylation, and we discuss both ongoing and future directions that will reveal the structural and mechanistic details of this essential eukaryotic cellular regulator.

Sortase A-mediated modification of the Streptococcus mutans transcriptome and virulence traits.

Sortase A contributes to adhesion and biofilm formation of Streptococcus mutans by anchoring surface proteins like P1 onto the cell wall, and few other functional characterization has been annotated to this protein and its coding gene srtA. In this study we investigated that whether srtA deletion would affect S. mutans virulence determinants in addition to adhesion and further explored whether these effects were caused due to changes in S. mutans genomic transcription. We used acid-killing assays, glycolytic rate assessments, and exopolysaccharide (EPS) formation tests to detect whether srtA deletion influenced S. mutans acid tolerance/production and glucan formation. Comparisons between RNA-sequencing data from both the exponential and stationary phases of UA159 and the srtA-deleted strain were made to determine the impact of srtA knockout on S. mutans genomic transcription. Results of our assays indicated that S. mutans aciduricity was enhanced in the srtA deleted strain when bacterial cells were directly subjected to pH 2.8, but the enhancement was repressed when the acid tolerance response was induced in advance. The srtA mutation strain exhibited reduced EPS formation in mature biofilms. SrtA deletion led to pleiotropic changes in the S. mutans transcriptome with a growth phase-dependent pattern. The affected genes mainly included those involved in aciduricity, carbohydrate transport, and EPS formation. It was concluded that S. mutans srtA exhibited multiple effects on the virulence traits of this pathogen, including acid tolerance and glucan formation, and that these alterations could be partially due to transcriptional changes upon loss of srtA.

A comprehensive in silico analysis of sortase superfamily.

Sortases are cysteine transpeptidases that assemble surface proteins and pili in their cell envelope. Encoded by all Gram-positive bacteria, few Gram-negative bacteria and archaea, sortases are currently divided into six classes (A-F). Due to the steep increase in bacterial genome data in recent years, the number of sortase homologues have also escalated rapidly. In this study, we used protein sequence similarity networks to explore the taxonomic diversity of sortases and also to evaluate the current classification of these enzymes. The resultant data suggest that sortase classes A, B, and D predominate in Firmicutes and classes E and F are enriched in Actinobacteria, whereas class C is distributed in both Firmicutes and Actinobacteria except Streptomyces family. Sortases were also observed in various Gram-negatives and euryarchaeota, which should be recognized as novel classes of sortases. Motif analysis around the catalytic cysteine was also performed and suggested that the residue at 2nd position from cysteine may help distinguish various sortase classes. Moreover, the sequence analysis indicated that the catalytic arginine is highly conserved in almost all classes except sortase F in which arginine is replaced by asparagine in Actinobacteria. Additionally, class A sortases showed higher structural variation as compared to other sortases, whereas inter-class comparisons suggested structures of class C and D2 exhibited best similarities. A better understanding of the residues highlighted in this study should be helpful in elucidating their roles in substrate binding and the sortase function, and successively could help in the development of strong sortase inhibitors.

Sortase-Dependent Proteins Promote Gastrointestinal Colonization by Enterococci.

The human gastrointestinal tract (GIT) is inhabited by a dense microbial community of symbionts. Enterococci are among the earliest members of this community and remain core members of the GIT microbiota throughout life. Enterococci have also recently emerged as opportunistic pathogens and major causes of nosocomial infections. Although recognized as a prerequisite for infection, colonization of the GIT by enterococci remains poorly understood. One way that bacteria adapt to dynamic ecosystems like the GIT is through the use of their surface proteins to sense and interact with components of their immediate environment. In Gram-positive bacteria, a subset of surface proteins relies on an enzyme called sortase for covalent attachment to the cell wall. Here, we show that the housekeeping sortase A (SrtA) enzyme promotes intestinal colonization by enterococci. Furthermore, we show that the enzymatic activity of SrtA is key to the ability of Enterococcus faecalis to bind mucin (a major component of the GIT mucus). We also report the GIT colonization phenotypes of E. faecalis mutants lacking selected sortase-dependent proteins (SDPs). Further examination of the mucin binding ability of these mutants suggests that adhesion to mucin contributes to intestinal colonization by E. faecalis.

Anchoring of LPXTG-Like Proteins to the Gram-Positive Cell Wall Envelope.

In Gram-positive bacteria, protein precursors with a signal peptide and a cell wall sorting signal (CWSS)-which begins with an LPXTG motif, followed by a hydrophobic domain and a tail of positively charged residues-are targeted to the cell envelope by a transpeptidase enzyme call sortase. Evolution and selective pressure gave rise to six classes of sortase, i.e., SrtA-F. Only class C sortases are capable of polymerizing substrates harboring the pilin motif and CWSS into protein polymers known as pili or fimbriae, whereas the others perform cell wall anchoring functions. Regardless of the products generated from these sortases, the basic principle of sortase-catalyzed transpeptidation is the same. It begins with the cleavage of the LPXTG motif, followed by the cross-linking of this cleaved product at the threonine residue to a nucleophile, i.e., an active amino group of the peptidoglycan stem peptide or the lysine residue of the pilin motif. This chapter will summarize the efforts to identify and characterize sortases and their associated pathways with emphasis on the cell wall anchoring function.

Staphylococcus aureus Sortase A-Mediated Incorporation of Peptides: Effect of Peptide Modification on Incorporation.

The endogenous Staphylococcus aureus sortase A (SrtA) transpeptidase covalently anchors cell wall-anchored (CWA) proteins equipped with a specific recognition motif (LPXTG) into the peptidoglycan layer of the staphylococcal cell wall. Previous in situ experiments have shown that SrtA is also able to incorporate exogenous, fluorescently labelled, synthetic substrates equipped with the LPXTG motif (K(FITC)LPETG-amide) into the bacterial cell wall, albeit at high concentrations of 500 µM to 1 mM. In the present study, we have evaluated the effect of substrate modification on the incorporation efficiency. This revealed that (i) by elongation of LPETG-amide with a sequence of positively charged amino acids, derived from the C-terminal domain of physiological SrtA substrates, the incorporation efficiency was increased by 20-fold at 10 µM, 100 µM and 250 µM; (ii) Substituting aspartic acid (E) for methionine increased the incorporation of the resulting K(FITC)LPMTG-amide approximately three times at all concentrations tested; (iii) conjugation of the lipid II binding antibiotic vancomycin to K(FITC)LPMTG-amide resulted in the same incorporation levels as K(FITC)LPETG-amide, but much more efficient at an impressive 500-fold lower substrate concentration. These newly developed synthetic substrates can potentially find broad applications in for example the in situ imaging of bacteria; the incorporation of antibody recruiting moieties; the targeted delivery and covalent incorporation of antimicrobial compounds into the bacterial cell wall.

Arginyltransferase suppresses cell tumorigenic potential and inversely correlates with metastases in human cancers.

Arginylation is an emerging post-translational modification mediated by arginyltransferase (ATE1) that is essential for mammalian embryogenesis and regulation of the cytoskeleton. Here, we discovered that Ate1-knockout (KO) embryonic fibroblasts exhibit tumorigenic properties, including abnormally rapid contact-independent growth, reduced ability to form cell-cell contacts and chromosomal aberrations. Ate1-KO fibroblasts can form large colonies in Matrigel and exhibit invasive behavior, unlike wild-type fibroblasts. Furthermore, Ate1-KO cells form tumors in subcutaneous xenograft assays in immunocompromised mice. Abnormal growth in these cells can be partially rescued by reintroduction of stably expressed specific Ate1 isoforms, which also reduce the ability of these cells to form tumors. Tumor array studies and bioinformatics analysis show that Ate1 is downregulated in several types of human cancer samples at the protein level, and that its transcription level inversely correlates with metastatic progression and patient survival. We conclude that Ate1-KO results in carcinogenic transformation of cultured fibroblasts, suggesting that in addition to its previously known activities Ate1 gene is essential for tumor suppression and also likely participates in suppression of metastatic growth.

Noninvasive imaging of immune responses.

At their margins, tumors often contain neutrophils, dendritic cells, and activated macrophages, which express class II MHC and CD11b products. The interplay between stromal cells, tumor cells, and migratory cells such as lymphocytes creates opportunities for noninvasive imaging of immune responses. We developed alpaca-derived antibody fragments specific for mouse class II MHC and CD11b products, expressed on the surface of a variety of myeloid cells. We validated these reagents by flow cytometry and two-photon microscopy to obtain images at cellular resolution. To enable noninvasive imaging of the targeted cell populations, we developed a method to site-specifically label VHHs [the variable domain (VH) of a camelid heavy-chain only antibody] with (18)F or (64)Cu. Radiolabeled VHHs rapidly cleared the circulation (t1/2 ≈ 20 min) and clearly visualized lymphoid organs. We used VHHs to explore the possibility of imaging inflammation in both xenogeneic and syngeneic tumor models, which resulted in detection of tumors with remarkable specificity. We also imaged the infiltration of myeloid cells upon injection of complete Freund's adjuvant. Both anti-class II MHC and anti-CD11b VHHs detected inflammation with excellent specificity. Given the ease of manufacture and labeling of VHHs, we believe that this method could transform the manner in which antitumor responses and/or infectious events may be tracked.

Molecular features of the sortase enzyme family.

Bacteria possess complex and varying cell walls with many surface exposed proteins. Sortases are responsible for the covalent attachment of specific proteins to the peptidoglycan of the cell wall of Gram-positive bacteria. Sortase A of Staphylococcus aureus, which is seen as the archetypal sortase, has been shown to be essential for pathogenesis and has therefore received much attention as a potential target for novel therapeutics. Being widely present in Gram-positive bacteria, it is likely that other Gram-positive pathogens also require sortases for their pathogenesis. Sortases have also been shown to be of significant use in a range of industrial applications. We review current knowledge of the sortase family in terms of their structures, functions and mechanisms and summarize work towards their use as antibacterial targets and microbiological tools.

Expression of Caenorhabditis elegans†PCS in the AtPCS1-deficient Arabidopsis thaliana†cad1-3 mutant separates the metal tolerance and non-host resistance functions of phytochelatin synthases.

Phytochelatin synthases (PCS) play key roles in plant metal tolerance. They synthesize small metal-binding peptides, phytochelatins, under conditions of metal excess. Respective mutants are strongly cadmium and arsenic hypersensitive. However, their ubiquitous presence and constitutive expression had long suggested a more general function of PCS besides metal detoxification. Indeed, phytochelatin synthase1 from Arabidopsis thaliana (AtPCS1) was later implicated in non-host resistance. The two different physiological functions may be attributable to the two distinct catalytic activities demonstrated for AtPCS1, that is the dipeptidyl transfer onto an acceptor molecule in phytochelatin synthesis, and the proteolytic deglycylation of glutathione conjugates. In order to test this hypothesis and to possibly separate the two biological roles, we expressed a phylogenetically distant PCS from Caenorhabditis elegans in an AtPCS1 mutant. We confirmed the involvement of AtPCS1 in non-host resistance by showing that plants lacking the functional gene develop a strong cell death phenotype when inoculated with the potato pathogen Phytophthora infestans. Furthermore, we found that the C. elegans gene rescues phytochelatin synthesis and cadmium tolerance, but not the defect in non-host resistance. This strongly suggests that the second enzymatic function of AtPCS1, which remains to be defined in detail, is underlying the plant immunity function.

Properties of a novel PBP2A protein homolog from Staphylococcus aureus strain LGA251 and its contribution to the ß-lactam-resistant phenotype.

Methicillin-resistant Staphylococcus aureus (MRSA) strains show strain-to-strain variation in resistance level, in genetic background, and also in the structure of the chromosomal cassette (SCCmec) that carries the resistance gene mecA. In contrast, strain-to-strain variation in the sequence of the mecA determinant was found to be much more limited among MRSA isolates examined so far. The first exception to this came with the recent identification of MRSA strain LGA251, which carries a new homolog of this gene together with regulatory elements mecI/mecR that also have novel, highly divergent structures. After cloning and purification in Escherichia coli, PBP2A(LGA), the protein product of the new mecA homolog, showed aberrant mobility in SDS-PAGE, structural instability and loss of activity at 37 °C, and a higher relative affinity for oxacillin as compared with cefoxitin. The mecA homolog free of its regulatory elements was cloned into a plasmid and introduced into the background of the ß-lactam-susceptible S. aureus strain COL-S. In this background, the mecA homolog expressed a high-level resistance to cefoxitin (MIC = 400 µg/ml) and a somewhat lower resistance to oxacillin (minimal inhibitory concentration = 200 µg/ml). Similar to PBP2A, the protein homolog PBP2A(LGA) was able to replace the essential function of the S. aureus PBP2 for growth. In contrast to PBP2A, PBP2A(LGA) did not depend on the transglycosylase activity of the native PBP2 for expression of high level resistance to oxacillin, suggesting that the PBP2A homolog may preferentially cooperate with a monofunctional transglycosylase as the alternative source of transglycosylase activity.

Protein ligation in living cells using sortase.

Sortagging is a versatile method for site-specific modification of proteins as applied to a variety of in vitro reactions. Here, we explore possibilities of adapting the sortase method for use in living cells. For intracellular sortagging, we employ the Ca²âº-independent sortase A transpeptidase (SrtA) from Streptococcus pyogenes. Substrate proteins were equipped with the C-terminal sortase-recognition motif (LPXTG); we used proteins with an N-terminal (oligo)glycine as nucleophiles. We show that sortase-dependent protein ligation can be achieved in Saccharomyces cerevisiae and in mammalian HEK293T cells, both in the cytosol and in the lumen of the endoplasmic reticulum (ER). ER luminal sortagging enables secretion of the reaction products, among which circular polypeptides. Protein ligation of substrate and nucleophile occurs within 30 min of translation. The versatility of the method is shown by protein ligation of multiple substrates with green fluorescent protein-based nucleophiles in different intracellular compartments.

Inhibition of glutaminyl cyclase attenuates cell migration modulated by monocyte chemoattractant proteins.

QC (glutaminyl cyclase) catalyses the formation of N-terminal pGlu (pyroglutamate) in peptides and proteins. pGlu formation in chemoattractants may participate in the regulation of macrophage activation and migration. However, a clear molecular mechanism for the regulation is lacking. The present study examines the role of QC-mediated pGlu formation on MCPs (monocyte chemoattractant proteins) in inflammation. We demonstrated in vitro the pGlu formation on MCPs by QC using MS. A potent QC inhibitor, PBD150, significantly reduced the N-terminal uncyclized-MCP-stimulated monocyte migration, whereas pGlu-containing MCP-induced cell migration was unaffected. QC small interfering RNA revealed a similar inhibitory effect. Lastly, we demonstrated that inhibiting QC can attenuate cell migration by lipopolysaccharide. These results strongly suggest that QC-catalysed N-terminal pGlu formation of MCPs is required for monocyte migration and provide new insights into the role of QC in the inflammation process. Our results also suggest that QC could be a drug target for some inflammatory disorders.

A novel bacterial resistance mechanism against human group IIA-secreted phospholipase A2: role of Streptococcus pyogenes sortase A.

Human group IIA-secreted phospholipase A(2) (sPLA(2)-IIA) is a bactericidal molecule important for the innate immune defense against Gram-positive bacteria. In this study, we analyzed its role in the host defense against Streptococcus pyogenes, a major human pathogen, and demonstrated that this bacterium has evolved a previously unidentified mechanism to resist killing by sPLA(2)-IIA. Analysis of a set of clinical isolates demonstrated that an ~500-fold higher concentration of sPLA(2)-IIA was required to kill S. pyogenes compared with strains of the group B Streptococcus, which previously were shown to be sensitive to sPLA(2)-IIA, indicating that S. pyogenes exhibits a high degree of resistance to sPLA(2)-IIA. We found that an S. pyogenes mutant lacking sortase A, a transpeptidase responsible for anchoring LPXTG proteins to the cell wall in Gram-positive bacteria, was significantly more sensitive (~30-fold) to sPLA(2)-IIA compared with the parental strain, indicating that one or more LPXTG surface proteins protect S. pyogenes against sPLA(2)-IIA. Importantly, using transgenic mice expressing human sPLA(2)-IIA, we showed that the sortase A-mediated sPLA(2)-IIA resistance mechanism in S. pyogenes also occurs in vivo. Moreover, in this mouse model, we also showed that human sPLA(2)-IIA is important for the defense against lethal S. pyogenes infection. Thus, we demonstrated a novel mechanism by which a pathogenic bacterium can evade the bactericidal action of sPLA(2)-IIA and we showed that sPLA(2)-IIA contributes to the host defense against S. pyogenes infection.

Staphylococcus aureus sortase A contributes to the Trojan horse mechanism of immune defense evasion with its intrinsic resistance to Cys184 oxidation.

Staphylococcus aureus is a Gram-positive bacterial pathogen that causes serious infections which have become increasingly difficult to treat due to antimicrobial resistance and natural virulence strategies. Bacterial sortase enzymes are important virulence factors and good targets for future antibiotic development. It has recently been shown that sortase enzymes are integral to bacterial survival of phagocytosis, an underappreciated, but vital, step in S. aureus pathogenesis. Of note, the reaction mechanism of sortases relies on a solvent-accessible cysteine for transpeptidation. Because of the common strategy of oxidative damage employed by professional phagocytes to kill pathogens, it is possible that this cysteine may be oxidized inside the phagosome, thereby inhibiting the enzyme. This study addresses this apparent paradox by assessing the ability of physiological reactive oxygen species, hydrogen peroxide and hypochlorite, to inhibit sortase A (SrtA) from S. aureus. Surprisingly, we found that SrtA is highly resistant to oxidative inhibition, both in vitro and in vivo. The mechanism of resistance to oxidative damage is likely mediated by maintaining a high reduction potential of the catalytic cysteine residue, Cys184. This is due to the unusual active site utilized by S. aureus SrtA, which employs a reverse protonation mechanism for transpeptidation, resulting in a high pK(a) as well as reduction potential for Cys184. The results of this study suggest that S. aureus SrtA is able to withstand the extreme conditions encountered in the phagosome and maintain function, contributing to survival of phagocytotic killing.

Functionality of sortase A in Lactococcus lactis.

Lactococcus lactis IL1403 harbors a putative sortase A (SrtA) and 11 putative sortase substrates that carry the canonical LPXTG signature of such substrates. We report here on the functionality of SrtA to anchor five LPXTG substrates to the cell wall, thus suggesting that SrtA is the housekeeping sortase in L. lactis IL1403.

The arginylation-dependent association of calreticulin with stress granules is regulated by calcium.

Post-translational modifications of proteins are important for the regulation of cell functions; one of these modifications is post-translational arginylation. In the present study, we show that cytoplasmic CRT (calreticulin) is arginylated by ATE1 (arginyl-tRNA protein transferase). We also show that a pool of CRT undergoes retrotranslocation from the ER (endoplasmic reticulum) to the cytosol, because in CRT-knockout cells transfected with full-length CRT (that has the signal peptide), cytoplasmic CRT appears as a consequence of its expression and processing in the ER. After the cleavage of the signal peptide, an N-terminal arginylatable residue is revealed prior to retrotranslocation to the cytoplasm where arginylation takes place. SGs (stress granules) from ATE1-knockout cells do not contain CRT, indicating that CRT arginylation is required for its association to SGs. Furthermore, R-CRT (arginylated CRT) in the cytoplasm associates with SGs in cells treated with several stressors that lead to a reduction of intracellular Ca2+ levels. However, in the presence of stressors that do not affect Ca2+ levels, R-CRT is not recruited to these loci despite the fact that SGs are formed, demonstrating Ca2+-dependent R-CRT association to SGs. We conclude that post-translational arginylation of retrotranslocated CRT, together with the decrease in intracellular Ca2+, promotes the association of CRT to SGs.

The Mycobacterium tuberculosis protein LdtMt2 is a nonclassical transpeptidase required for virulence and resistance to amoxicillin.

The peptidoglycan layer is a vital component of the bacterial cell wall. The existing paradigm describes the peptidoglycan network as a static structure that is cross-linked predominantly by 4-->3 transpeptide linkages. However, the nonclassical 3-->3 linkages predominate the transpeptide networking of the peptidoglycan layer of nonreplicating Mycobacterium tuberculosis. The molecular basis of these linkages and their role in the physiology of the peptidoglycan layer, virulence and susceptibility of M. tuberculosis to drugs remain undefined. Here we identify MT2594 as an L,D-transpeptidase that generates 3-->3 linkages in M. tuberculosis. We show that the loss of this protein leads to altered colony morphology, loss of virulence and increased susceptibility to amoxicillin-clavulanate during the chronic phase of infection. This suggests that 3-->3 cross-linking is vital to the physiology of the peptidoglycan layer. Although a functional homolog exists, expression of ldtMt2 is dominant throughout the growth phases of M. tuberculosis. 4-->3 transpeptide linkages are targeted by one of the most widely used classes of antibacterial drugs in human clinical use today, beta-lactams. Recently, meropenem-clavulanate was shown to be effective against drug-resistant M. tuberculosis. Our study suggests that a combination of L,D-transpeptidase and beta-lactamase inhibitors could effectively target persisting bacilli during the chronic phase of tuberculosis.