Posttranslational Modification Enzymes Research Articles

In vitro catalytic activity of N-terminal and C-terminal domains in NukM, the post-translational modification enzyme of nukacin ISK-1

Lantibiotics are antibacterial peptides containing unique thioether cross-links termed lanthionine and methyllanthionine. NukM, the modifying enzyme of nukacin ISK-1, which is produced by Staphylococcus warneri ISK-1, catalyzes the dehydration of specific Ser/Thr residues in a precursor peptide, followed by conjugative addition of intramolecular Cys to dehydrated residues to generate a cyclic structure. By contrast, the precursor peptide of nisin is modified by 2 enzymes, NisB and NisC, which mediate dehydration and cyclization, respectively. While the C-terminal domain of NukM is homologous to NisC, the N-terminal domain has no homology with other known proteins. We expressed and characterized the N- and C-terminal domains of NukM, NukMN, and NukMC, separately. In vitro reconstitution revealed that full-length NukM fully modified the substrate peptide NukA. NukMN partially phosphorylated, dehydrated, and cyclized NukA. By contrast, NukMC did not catalyze dehydration, phosphorylation, or cyclization reactions. Interaction studies using surface plasmon resonance analysis indicated that NukM and NukMN can bind NukA with high affinity, whereas NukMC has low substrate-recognition activity. These results suggest that NukMN is mainly responsible for substrate recognition and dehydration and that the whole NukM structure, including the C-terminal domain, is required for the complete modification of NukA. To the best of our knowledge, this is the first report providing insights into the in vitro catalytic activity of individual domains of a LanM-type modification enzyme.

Abstract 1154: Age of type I collagen is critical for regulation of HT-1080 cell proliferation in 3D matrices

Abstract The tumor microenvironnement consists of cells, soluble factors, signaling molecules, extracellular matrix (EM), and mechanical cues that can promote neoplastic transformation, support tumor growth and invasion, and foster therapeutic resistance. In addition to provide a physical barrier against invasion, type I collagen, the most abundant protein in EM, is also known to play a role in tumor cell proliferation (1). DeClerck et al. (2) has reported a direct inhibitory effect of fibrillar type I collagen on tumor cell proliferation when compared to the monomeric one. Type I collagen is a life-long protein (half-life 15 yrs) susceptible to undergo non enzymatic post-translational modifications such as production of Advanced Glycation End Products (AGEs). These modifications are able to influence the behavior of tumor cells. Here, we investigated whether regulation of HT-1080 cell proliferation is a consequence of type I collagen aging in terms of fibrillar state of the protein (2,3), or of the level of AGEs, also known to stimulate cell proliferation (4). Rat tail type I collagen were prepared from adult (2 months) and old (2 years) animals. A significant increase in AGEs has been observed in collagen extracted from old rats, compared to the adult. Under 3D growth condition, HT-1080 cells proliferate rapidly in old type I collagen relative to the adult one. This effect was not observed in 2D coating culture. The low rate of proliferation in adult collagen is accompanied by a downregulation of ERK1/2 activation, and an upregulation of p21, the inhibitor of cell cycle progression. This age-dependent cell proliferation regulatory effect does not involve α2β1 integrins. Neither Pi3K/Akt nor JNK pathways were involved in this process. Accumulating evidence suggest that Discoidin Domain Receptor 2 (DDR2) is a Receptor Tyrosine Kinase (RTK) with the unique ability among RTKs to respond to fibrillar collagen (5). To determine whether a similar response is elicited by DDR2 in both collagens, DDR2 was immunoprecipitated from cell lysates, and phosphorylated DDR2 was detected by immunoblotting. High level of DDR2 phosphorylation was observed after 3 days culture in adult collagen compared to the old one, whereas DDR2 expression remains unchanged in the presence of both collagens. Moreover, the DDR2 kinase function inhibitor, nilotinib, restored cell proliferation in adult collagen to a level similar to that observed in the old one. Taken together, these data establish a role for DDR2 in critical events during restriction of tumor cell proliferation induced by adult type I collagen.

Microtubule chemical complexity: mechanism of tubulin modification enzymes

Tubulin is subject to an abundant and diverse set of post-translational modifications that include phosphorylation, acetylation, poly-glutamylation, poly-glycylation and tyrosination. The highest density and variety of post-translational modifications are found in especially complex microtubule arrays like those of neurons or cilia. Not surprisingly, tubulin modification enzymes have been linked to human diseases including cancers and neurodegenerative disorders. I will present recent data from my lab on the mechanism of action of two tubulin modification enzymes that illustrate two divergent paradigms of tubulin recognition. Tubulin tyrosine ligase (TTL) adds a C-terminal Tyr to the exposed C-terminus of alpha-tubulin as part of a tyrosination/detyrosination cycle present in most eukaryotic cells. We solved the first crystal structure of tubulin tyrosine ligase that revealed how the TTL scaffold supported the expansion of the repertory of tubulin post-translational modification enzymes of the TTL like family that recognize either alpha- or beta-tubulin C-terminal tails. In addition to modifying tubulin, TTL also prevents tubulin from incorporating into microtubules by recognizing a tubulin dimer interface that would otherwise be involved in microtubule lattice interactions. I will also present recent work from my group on the structure and mechanism of action of tubulin acetyltransferase (TAT). TAT acetylates Lys-40 on alpha-tubulin in the microtubule lumen. We solved the 2.7Å structure of TAT bound to its ac-coA substrate as well as the 2.45Å structure of a catalytic inactive TAT mutant that reveals a domain swapped dimer in which the functionally essential N-terminus shows evidence of unprecedented structural plasticity. Implications for catalysis and microtubule stimulation of TAT activity will be discussed.

Proteomics of citrullination in cardiovascular disease

Citrullination is an enzymatic posttranslational modification (PTM), which has become a topic of recent research due to its involvement in various physiologic and pathologic processes. This review will focus primarily on the cardiovascular pathology associated to date with citrullination, including myocardial citrullination as well as the potential role of citrullination in atherosclerosis as a driver inflammation, especially in patients with rheumatoid arthritis (RA). There is extensive citrullination within normal and RA myocardium as well as the atherosclerotic plaque; and increased levels of antibodies to citrullinated proteins have been associated with increased cardiovascular burden in patients with RA. Given robust citrullination in both RA as well as non-RA patients, there also exists the potential for protein citrullination to contribute to cardiovascular pathology in the general population. However, to investigate this possibility will require development of improved biochemical and proteomic tools for the study of protein citrullination. The remainder of this review will discuss current and developing methodologies to study protein citrullination and discuss their applicability for the analysis of complex samples. The ability to identify and quantify citrullinated protein is a key to understanding the role of this PTM. Methodologies and limitations of current technology for the identification of citrullination are discussed.

Serum specific IgG response to toluene diisocyanate-tissue transglutaminase conjugate in toluene diisocyanate–induced occupational asthmatics

BackgroundTissue transglutaminase (tTG) is a post-translational modifying enzyme located in airway epithelial cells. A potential contribution of serum specific IgG (sIgG) to tTG in airway inflammation of toluene diisocyanate (TDI)-induced occupational asthma (OA) has been suggested. ObjectiveTo prepare a TDI-tTG conjugate and detect serum specific antibodies in sera of patients with TDI-OA to understand this mechanism. MethodsNinety-nine patients with TDI-OA, 76 asymptomatic exposed controls, 208 patients with non-OA, and 74 unexposed controls were enrolled for this study. The TDI-tTG conjugate was prepared and confirmed by a native gel. Serum sIgG and/or sIgE antibodies to tTG, TDI-tTG, TDI conjugated to human serum albumin, cytokeratin 19, and serum cytokine levels, such as interleukin-8, transforming growth factor-β1, and tissue inhibitor of metalloproteinase-1, were measured by enzyme-linked immunosorbent assay. The level of interleukin-8 produced from airway epithelial cells (A549) treated with tTG was evaluated to investigate the inflammatory effect of tTG and TDI-tTG. ResultsIn the TDI-OA group, the prevalence of serum sIgG to TDI-tTG (17.2%) was higher than that of sIgG to tTG (11.1%), which were significantly higher than those of the 3 control groups (P < .05 for all groups). TDI-exposed subjects with high levels of serum sIgG to TDI-tTG had a high prevalence of sIgG to cytokeratin 19 and higher serum levels of transforming growth factor-β1 and tissue inhibitor of metalloproteinase-1. The tTG and TDI-tTG dose-dependently increased interleukin-8 production from A549 cells. ConclusionThese findings suggest that TDI exposure in the workplace binds to tTG to form a conjugate that can induce serum sIgG antibody production, airway inflammation, and airway remodeling in patients with TDI-OA.

Biogenesis of D-amino acid containing peptides/proteins: where, when and how?

Peptides and proteins are chiral molecules with their structure determined by the composition and configuration of the amino acids constituting them. Natural amino acids (except glycine) display two chiral types (l- and d-enantiomers). For example, the presence of octopine, a derivative of l-arginine and d-alanine in octopus, or peptidyl poly-d-glutamic acid in a bacterial cell wall was demonstrated in the 1920s and 1930s, respectively. Nevertheless, an old dogma in biology was that proteins (in a strict sense) are composed of amino acids in the l-configuration exclusively, until a d-alanyl residue was reported in a frog skin opioid peptide in the early 1980s, and since, numerous d-amino acid containing peptides (DAACPs) have been discovered in multicellular organisms. Several hypotheses may be formulated to explain the origin of a d-residue in the peptide/protein chain. It may result from different mechanisms such as incorporation of a d-amino acid, non-enzymatic racemisation associated with ageing or diseases and enzymatic posttranslational modification. In the last case, the DAACPs are synthesised via a ribosome-dependent manner, and a normal codon for l-amino acid is present in the mRNA at the position where the d-residue is processed in the mature peptide by peptidyl aminoacyl l-d isomerisation, a peculiar and subtle posttranslational modification. In this review, the different pathways of biogenesis of DAACPs not only in bacteria but also in multicellular organisms are discussed, along with the description of the cellular specificity, the enzyme specificity and the substrate specificity of peptidyl aminoacyl l-d isomerisation.

A Substrate Preference for the Rough Endoplasmic Reticulum Resident Protein FKBP22 during Collagen Biosynthesis

The biosynthesis of collagens occurs in the rough endoplasmic reticulum and requires a large numbers of molecular chaperones, foldases, and post-translational modification enzymes. Collagens contain a large number of proline residues that are post-translationally modified to 3-hydroxyproline or 4-hydroxyproline, and the rate-limiting step in formation of the triple helix is the cis-trans isomerization of peptidyl-proline bonds. This step is catalyzed by peptidyl-prolyl cis-trans isomerases. There are seven peptidyl-prolyl cis-trans isomerases in the rER, and so far, two of these enzymes, cyclophilin B and FKBP65, have been shown to be involved in collagen biosynthesis. The absence of either cyclophilin B or FKBP65 leads to a recessive form of osteogenesis imperfecta. The absence of FKBP22 leads to a kyphoscoliotic type of Ehlers-Danlos syndrome (EDS), and this type of EDS is classified as EDS type VI, which can also be caused by a deficiency in lysyl-hydroxylase 1. However, the lack of FKBP22 shows a wider spectrum of clinical phenotypes than the absence of lysyl-hydroxylase 1 and additionally includes myopathy, hearing loss, and aortic rupture. Here we show that FKBP22 catalyzes the folding of type III collagen and interacts with type III collagen, type VI collagen, and type X collagen, but not with type I collagen, type II collagen, or type V collagen. These restrictive interactions might help explain the broader phenotype observed in patients that lack FKBP22.

A new antimicrobial substance produced by Staphylococcus pasteuri isolated from vegetables

Staphylococcus aureus is important food-borne pathogen in agricultural products consumed in a fresh state. The antimicrobial activities of staphylococci isolated from vegetables in Korea against S. aureus were investigated. Coagulase-negative staphylococci (CNS) isolates (12) with antimicrobial activities against S. aureus were identified as S. xylosus, pasteuri, and epidermidis. CNS isolates exhibited antimicrobial activities against Gram-positive bacteria. Inactivation of S. pasteuri antimicrobial activity by proteases suggested that the substances evaluated were antimicrobial peptides or bacteriocins. None of the S. pasteuri isolates possessed homologous DNA fragments known to be bacteriocin genes, except for nukacin. The nukM gene that encodes a post-translational modification enzyme in the nukacin gene cluster was not detected in S. pasteuri isolates. S. pasteuri probably produces one or more new antimicrobial substances.

Combinatorial assembly of large biochemical pathways into yeast chromosomes for improved production of value-added compounds.

Saccharomyces cerevisiae as a eukaryotic organism is particularly suitable as microbial cell factory because it has interesting features such as membrane environments for supporting membrane-associated enzymes and its capability for post-translational modifications of enzymes from plants. However, S. cerevisiae does not readily express polycistronic transcriptional units, which represents a significant challenge for constructing large biochemical pathways in budding yeast. In the present study, we developed a novel approach for rapid construction of large biochemical pathways into yeast chromosomes. Our approach takes advantage of antibiotic selection for combinatorial assembly of large pathways into the δ-sites of retrotransposon elements of yeast chromosomes. As proof-of-principle, a five-gene isobutanol pathway and an eight-gene mevalonate pathway were successfully assembled into yeast chromosomes in one-step fashion. To our knowledge, this is the first report to exploit δ-integration coupled with antibiotic selection for rapid assembly of large biochemical pathways in budding yeast. We envision our new approach could serve as a generalized technique for large pathway construction in yeast-a method that would be of significant interest to the synthetic biology community.

Identification of Ligand Specificity Determinants in Lantibiotic Bovicin HJ50 and the Receptor BovK, a Multitransmembrane Histidine Kinase

Lantibiotic bovicin HJ50 is produced by Streptococcus bovis HJ50 and acts as the extracellular signal to autoregulate its own biosynthesis through BovK/R two-component system. Bovicin HJ50 shows a linear N-terminal and glubolar C-terminal structure, and the sensor histidine kinase BovK contains eight transmembrane segments lacking any extensive surface-exposed sensory domain. The signal recognition mechanism between bovicin HJ50 and BovK is still unknown. We performed saturated alanine scanning mutagenesis and other amino acid substitutions on bovicin HJ50 using a semi-in vitro biosynthesis. Results of the mutants inducing activities indicated that several charged and hydrophobic amino acids in ring B of bovicin HJ50, as well as two glycines were key residues to recognize BovK. Circular dichroism analyses indicated that both glycines contributed to bovicin HJ50 structural changes in the membrane. Biotin-labeled bovicin HJ50 could interact with the N-terminal sensor of BovK, and several charged residues and a conserved hydrophobic region in the N-terminal portion of BovK sensor domain were important for interacting with the signal bovicin HJ50. By combining the results, we suggested a mechanism of bovicin HJ50 recognizing and activating BovK mainly through electrostatic and hydrophobic interactions.

Studying post-translational modifications with protein interaction networks

At least 46 interactome studies, broad at proteome scale or biologically more focused, have together mapped about 75,000 human protein-protein interactions (PPIs). Many of the studies addressed local interactome data paucity analyzing specific homeostatic and regulatory systems, with recent focus demonstrating the involvement of post-translational protein modification (PTM) enzyme families in a wide range of cellular functions. These datasets provided insight into binding mechanisms, the dynamic modularity of complexes or delineated combinatorial enzymatic cascades. Furthermore, the combined study of PPI and PTM dynamics has begun to reveal conditional rewiring of molecular networks through PTM-mediated recognition events. Taken together these studies highlight the utility of local and global interaction networks to functionally prioritize the many changing PTMs mapped in human cells.

Peptide-Templated Gold Nanocluster Beacon as a Sensitive, Label-Free Sensor for Protein Post-translational Modification Enzymes

Protein post-translational modifications (PTMs), which are chemical modifications and most often regulated by enzymes, play key roles in functional proteomics. Detection of PTM enzymes, thus, is critical in the study of cell functioning and development of diagnostic and therapeutic tools. Herein, we develop a simple peptide-templated method to direct rapid synthesis of highly fluorescent gold nanoclusters (AuNCs) and interrogate the effect of enzymatic modifications on their luminescence. A new finding is that enzymes are able to exert chemical modifications on the peptide-templated AuNCs and quench their fluorescence, which furnishes the development of a real-time and label-free sensing strategy for PTM enzymes. Two PTM enzymes, histone deacetylase 1 and protein kinase A, have been employed to demonstrate the feasibility of this enzyme-responsive fluorescent nanocluster beacon. The results reveal that the AuNCs' fluorescence can be dynamically decreased with increasing concentration of the enzymes, and subpicomolar detection limits are readily achieved for both enzymes. The developed strategy can thus offer a useful, label-free biosensor platform for the detection of protein-modifying enzymes and their inhibitors in biomedical applications.

Three isozymes of peptidylarginine deiminase in the chicken: Molecular cloning, characterization, and tissue distribution

Peptidylarginine deiminase (PAD; EC 3.5.3.15) is a post-translational modification enzyme that catalyzes the conversion of protein-bound arginine to citrulline (deimination) in a calcium ion dependent manner. Although PADI genes are widely conserved among vertebrates, their function in the chicken is poorly understood. Here, we cloned and sequenced three chicken PADI cDNAs and analyzed the expression of their proteins in various tissues. Immunoblotting analysis showed that chicken PAD1 and PAD3 were present in cells of several central neuron system tissues including the retina; the chicken PAD2 protein was not detected in any tissue. We expressed recombinant chicken PADs in insect cells and characterized their enzymatic properties. The chicken PAD1 and PAD3 recombinant proteins required calcium ions as an essential cofactor for their catalytic activity. The two recombinant proteins showed similar substrate specificities toward synthetic arginine derivatives. By contrast to them, chicken PAD2 did not show any activity. We found that one of the conserved active centers in mammalian PADs had been altered in chicken PAD2; we prepared a reverse mutant but we did not detect an activity. We conclude that chicken PAD1 and PAD3 might play specific roles in the nervous system, but that chicken PAD2 might not be functional under normal physiological conditions.

ChemInform Abstract: Ribosomally Synthesized and Post‐Translationally Modified Peptide Natural Products: New Insights into the Role of Leader and Core Peptides During Biosynthesis

AbstractReview: 164 refs.

Dissection of Cdk1–cyclin complexes in vivo

Cyclin-dependent kinases (Cdks) are regulatory enzymes with temporal and spatial selectivity for their protein substrates that are governed by cell cycle-regulated cyclin subunits. Specific cyclin-Cdk complexes bind to and phosphorylate target proteins, coupling their activity to cell cycle states. The identification of specific cyclin-Cdk substrates is challenging and so far, has largely been achieved through indirect correlation or use of in vitro techniques. Here, we use a protein-fragment complementation assay based on the optimized yeast cytosine deaminase to systematically identify candidate substrates of budding yeast Saccharomyces cerevisiae Cdk1 and show dependency on one or more regulatory cyclins. We identified known and candidate cyclin dependencies for many predicted protein kinase Cdk1 targets and showed elusory Clb3-Cdk1-specific phosphorylation of γ-tubulin, thus establishing the timing of this event in controlling assembly of the mitotic spindle. Our strategy can be generally applied to identify substrates and accessory subunits of multisubunit protein complexes.

Ribosomally Synthesized and Post‐Translationally Modified Peptide Natural Products: New Insights into the Role of Leader and Core Peptides during Biosynthesis

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with a high degree of structural diversity and a wide variety of bioactivities. Understanding the biosynthetic machinery of these RiPPs will benefit the discovery and development of new molecules with potential pharmaceutical applications. In this Concept article, we discuss the features of the biosynthetic pathways to different RiPP classes, and propose mechanisms regarding recognition of the precursor peptide by the post-translational modification enzymes. We propose that the leader peptides function as allosteric regulators that bind the active form of the biosynthetic enzymes in a conformational selection process. We also speculate how enzymes that generate polycyclic products of defined topologies may have been selected for during evolution.

Hyper-O-GlcNAcylation Is Anti-apoptotic and Maintains Constitutive NF-κB Activity in Pancreatic Cancer Cells

Cancer cell metabolic reprogramming includes a shift in energy production from oxidative phosphorylation to less efficient glycolysis even in the presence of oxygen (Warburg effect) and use of glutamine for increased biosynthetic needs. This necessitates greatly increased glucose and glutamine uptake, both of which enter the hexosamine biosynthetic pathway (HBP). The HBP end product UDP-N-acetylglucosamine (UDP-GlcNAc) is used in enzymatic post-translational modification of many cytosolic and nuclear proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). Here, we observed increased HBP flux and hyper-O-GlcNAcylation in human pancreatic ductal adenocarcinoma (PDAC). PDAC hyper-O-GlcNAcylation was associated with elevation of OGT and reduction of the enzyme that removes O-GlcNAc (OGA). Reducing hyper-O-GlcNAcylation had no effect on non-transformed pancreatic epithelial cell growth, but inhibited PDAC cell proliferation, anchorage-independent growth, orthotopic tumor growth, and triggered apoptosis. PDAC is supported by oncogenic NF-κB transcriptional activity. The NF-κB p65 subunit and upstream kinases IKKα/IKKβ were O-GlcNAcylated in PDAC. Reducing hyper-O-GlcNAcylation decreased PDAC cell p65 activating phosphorylation (S536), nuclear translocation, NF-κB transcriptional activity, and target gene expression. Conversely, mimicking PDAC hyper-O-GlcNAcylation through pharmacological inhibition of OGA suppressed suspension culture-induced apoptosis and increased IKKα and p65 O-GlcNAcylation, accompanied by activation of NF-κB signaling. Finally, reducing p65 O-GlcNAcylation specifically by mutating two p65 O-GlcNAc sites (T322A and T352A) attenuated the induction of PDAC cell anchorage-independent growth. Our data indicate that hyper-O-GlcNAcylation is anti-apoptotic and contributes to NF-κB oncogenic activation in PDAC.

Biochemical dissection of bacterial virulence and macrophage innate immunity

Many bacterial pathogens use a type III secretion system to inject virulent effector proteins into host cells. These effectors often use sophisticated biochemical strategies to manipulate host signal transduction pathway. I will present our work on three bacterial effector families, each of which defines a novel enzymatic posttranslational modification, and their functions in infection. The OspF effector from Shigella flexneri employs a MAPK phosphothreonine lyase activity to block host MAPK signaling and IL‐8 production. The CHBP/Cif family of effectors from Burkholderia pseudomallei and Enteropathogenic E. coli (EPEC) catalyze deamidation of Gln‐40 in ubiquitin and NEDD8, inactivating host ubiquitin pathway and many related cellular processes. NleE from EPEC and Salmonella typhimurium carries out a novel cysteine methylation modification on key ubiquitin‐chain sensory proteins in host NF‐kB pathway, blocking NF‐kB‐mediated inflammatory response during infection. On the host side, the inflammasome complex is believed to be important for macrophage innate immune defense against bacterial infection. I will also discuss our recent identification and characterization of the NAIP family of NOD‐like proteins (NLRs) that serves as inflammasome receptors for bacterial flagellin and also the type III secretion apparatus, which plays an important role in restricting various bacterial infections.

Peptidylarginine Deiminase and Citrullination: Potential Therapeutic Targets for Inflammatory Diseases

The multiple post-translational modifications of proteins display specific gain- or loss-of-function under normal and abnormal conditions. These modifications are precisely regulated by post-translational modification enzymes. The altered molecular status perturbs the pattern of gene expression and decides on a direction to signal transduction cascades as well as intrinsic properties of the proteins. Ultimately, it strictly maintains intracellular environment or results in disease manifestations. Recently, it has become that enzyme-dependent modification of arginine residue to citrulline exerts an important role in the induction of autoimmunity including rheumatoid arthritis, multiple sclerosis, and cancer. The modification of arginine residue to citrulline on proteins is called citrullination or deimination and is regulated by the calcium-dependent enzyme peptidylarginine deiminase (PAD). Now many effective PAD inhibitors (for example, Cl-amidine) have developed that ameliorates disease phenotypes. In this review, we discuss crucial roles of PAD enzyme and citrullination, the effectiveness of PAD inhibitors, and the implication in pathology.

Kinetic isotope effects for studying post-translational modifying enzymes

The ongoing development of new experimental approaches for the measurement of isotope effects is improving our understanding of the physical and chemical changes that occur during biological catalysis. Biological catalysis involves numerous steps that include binding, conformational changes, chemical catalysis and product release. The critical points on the free energy surface for biologically catalyzed reactions include all bound intermediates and the intervening transition states. Isotope effects can be used to investigate both intermediate (equilibrium isotope effects) and transition state (kinetic isotope effects) structures along the reaction coordinate. This review details new techniques for measuring isotope effects and provides several examples of their use in solving transition state structures for post-translational modifying enzymes.