Posttranslational Modification Enzymes Research Articles

Mathermycin, a Lantibiotic from the Marine Actinomycete Marinactinospora thermotolerans SCSIO 00652.

Lantibiotics are antimicrobial peptides belonging to the family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) and feature thioether linkages in their structures. In this study, we identified the biosynthetic gene cluster of a cinnamycin analog, named mathermycin, from Marinactinospora thermotolerans SCSIO 00652 and reconstituted its biosynthesis in Streptomyces lividans and Escherichia coli Key posttranslational modification enzymes of mathermycin were characterized. Mathermycin exhibited antimicrobial activity and therefore represents an example of cinnamycin-like lantibiotics from Marinactinospora species.IMPORTANCE The discovery of new antimicrobial compounds that can be used as potential drugs is in urgent need due to increasing bacterial resistance to current antibiotics. Lantibiotics are important antimicrobial compounds that have found applications in both the clinic setting and food industry. We report here the discovery of a new lantibiotic, mathermycin, from a marine-derived Marinactinospora thermotolerans strain and elucidation of its biosynthesis. We also demonstrate that mathermycin possesses antimicrobial activity toward a Bacillus strain.

Abstract 4096: Transglutaminase 2(TG2) induces the intrinsic EGFR-TKI resistance in EGFR-mutant NSCLC

Abstract The non-small cell lung cancer (NSCLC) patients with EGFR-sensitive mutations can be therapeutically targeted by EGFR tyrosine kinase inhibitors (EGFR-TKI), such as erlotinib. Unfortunately, even though the intrinsic EGFR-TKI resistant patients harboring the EGFR-sensitizing mutation, a part of those are still resistant to EGFR-TKI. And, it has been reported that both PTEN loss and NF-kB activation contribute to intrinsic EGFR-TKI resistance in EGFR-mutant lung cancer. Transglutaminse 2 (TG2) is post-translational modification enzyme and known to induce degradation of tumor suppressors including PTEN and IκB-α with peptide cross-linking activity. Because TG2 was known as a regulator of PTEN and IκB-α (NF-κB inhibitor) level in cytosol, we have explored if TG2 can be another key regulator to the intrinsic resistance of EGFR-TKI in the intrinsic EGFR-TKI resistant NSCLC cell. In NSCLC with EGFR-sensitive mutations, we first found that higher TG2 expression level and lower PTEN and IκB-α expression levels in the intrinsic EGFR-TKI resistant NSCLC compare with EGFR-TKI sensitive NSCLC. TG2 stably expressing EGFR-TKI sensitive NSCLC cells harboring EGFR mutations showed reduction of both PTEN and IκB-α and exhibited EGFR-TKI resistance. In reverse, TG2 stably downregulated H1650, intrinsic EGFR-TKI resistant NSCLC cell harboring EGFR sensitive mutation, restored EGFR-TKI sensitivity via PTEN and IκB-α restoration by TG2 loss. Xenograft study using TG2 overexpressed and downregulated NSCLC cells also verified our in vitro results. Therefore, we have demonstrated that TG2 elicits the intrinsic EGFR-TKI resistance via PTEN loss and activation of NF-κB pathway. These results suggest that TG2 may be a useful target for overcoming the intrinsic EGFR-TKI resistance of NSCLC harboring EGFR sensitive mutations. Citation Format: Junyoung Choi, Hannah Yang, Sang-We Kim, Kyung Hae Jung, Kang-Seo Park, Dae Ho Lee. Transglutaminase 2(TG2) induces the intrinsic EGFR-TKI resistance in EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4096. doi:10.1158/1538-7445.AM2017-4096

Listeriolysin S Is a Streptolysin S-Like Virulence Factor That Targets Exclusively Prokaryotic Cells In Vivo.

ABSTRACTStreptolysin S (SLS)-like virulence factors from clinically relevant Gram-positive pathogens have been proposed to behave as potent cytotoxins, playing key roles in tissue infection. Listeriolysin S (LLS) is an SLS-like hemolysin/bacteriocin present among Listeria monocytogenes strains responsible for human listeriosis outbreaks. As LLS cytotoxic activity has been associated with virulence, we investigated the LLS-specific contribution to host tissue infection. Surprisingly, we first show that LLS causes only weak red blood cell (RBC) hemolysis in vitro and neither confers resistance to phagocytic killing nor favors survival of L. monocytogenes within the blood cells or in the extracellular space (in the plasma). We reveal that LLS does not elicit specific immune responses, is not cytotoxic for eukaryotic cells, and does not impact cell infection by L. monocytogenes. Using in vitro cell infection systems and a murine intravenous infection model, we actually demonstrate that LLS expression is undetectable during infection of cells and murine inner organs. Importantly, upon intravenous animal inoculation, L. monocytogenes is found in the gastrointestinal system, and only in this environment LLS expression is detected in vivo. Finally, we confirm that LLS production is associated with destruction of target bacteria. Our results demonstrate therefore that LLS does not contribute to L. monocytogenes tissue injury and virulence in inner host organs as previously reported. Moreover, we describe that LlsB, a putative posttranslational modification enzyme encoded in the LLS operon, is necessary for murine inner organ colonization. Overall, we demonstrate that LLS is the first SLS-like virulence factor targeting exclusively prokaryotic cells during in vivo infections.

Transglutaminases in Dysbiosis As Potential Environmental Drivers of Autoimmunity.

Protein-glutamine γ-glutamyltransferases (transglutaminases, Tgs) belong to the class of transferases. They catalyze the formation of an isopeptide bond between the acyl group at the end of the side chain of protein- or peptide-bound glutamine residues and the first order 𝜀-amine groups of protein- or peptide-bound lysine. The Tgs are considered to be universal protein cross-linkers, and they play an essential role in a number of human diseases. In this review, we discuss mainly the bacterial Tgs in terms of the functionality of the enzymes and a potential role they may play in bacterial survival. Since microbial transglutaminases (mTgs) are functionally similar to the human homologs, they may be involved in the human disease provocation. We suggest here a potential involvement of Tgs in the pathologies such as autoimmune diseases. In this hypothesis, the endogenous mTgs that are secreted by the gut microbiota, especially in a dysbiotic configuration, are potential drivers of systemic autoimmunity, via the enzymatic posttranslational modification of peptides in the gut lumen. These mTg activities directed toward cross-linking of naïve proteins can potentially generate neo-epitopes that are not only immunogenic but may also activate some immune response cascades leading to the pathological autoimmune processes.

Biochemical Analysis of Histone Succinylation.

Posttranslational modification (PTM) of proteins is used to regulate protein activity and stability. Histone PTMs are regarded as some of the most important, as they can directly regulate gene expression through chromatin reorganization. Recently, histone proteins were found to undergo succinylation, adding to other well-known PTMs such as acetylation, methylation, and phosphorylation. However, there is little information regarding the enzyme which catalyzes histone lysine succinylation. In fact, it is unclear whether this reaction is enzymatic. In this study, we tested histone succinylation activity in vitro using cell nuclear extracts of HepG2 cells. Although whole nuclear extracts did not show histone succinylation activity, we found that an SP 1.0 M KCl fraction of nuclear extracts indeed had such activity. These data offer the first direct evidence that histone succinylation is an enzymatic PTM as are other histone codes in the nucleus.

Tissue transglutaminase (TG2) and mitochondrial function and dysfunction.

Mitochondria are the cell's power plant to satisfy the energy demands. However, dysfunctional mitochondria can cause overproduction of reactive oxygen species (ROS), oxidative stress, and alteration of calcium homeostasis, which are the hallmarks of mitochondrial diseases. Under prolong oxidative stress, repeated cytosolic calcium elevations even only transiently, can lead to activation of some enzymes. One calcium-activated enzyme with demonstrated pathophysiological important in mitochondrial disease is tissue transglutaminase (TG2). TG2 is known as a post-translational modification (PTM) enzyme that is induced by oxidative stress. Compared to other types of PTMs, the physiological significance of TG2 mediated PTM is just beginning to be understood. Once activated, TG2 can modulate transcription, inactivate metabolic enzymes, and cause aggregation of critical proteins. Recent data indicate that TG2's activity not only can modulate the assembly of respiratory chain complexes but can also modulate the transcription of critical genes including PGC-1alpha and cytochrome C that are important for function and biogenesis of mitochondria. Here, we summarize dysfunctional mitochondria in diseases such as in neurodegenerative disorders can modulate TG2's activity and function. TG2 is also important for normal function of mitochondria.

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)-PKMT-substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (SN2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MS-based method to measure KIEs and binding isotope effects (BIEs) of the cofactor S-adenosyl-l-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic 13C KIE of 1.04, an inverse intrinsic α-secondary CD3 KIE of 0.90, and a small but statistically significant inverse CD3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical SN2 transition state with the C-N and C-S distances of 2.35-2.40 Å and 2.00-2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steady-state kinetics with the SAM analog Se-adenosyl-l-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.

Induction of peptidylarginine deiminase 2 and 3 by dibutyryl cAMP via cAMP-PKA signaling in human astrocytoma U-251MG cells.

Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that citrullinate (deiminate) protein arginine residues in a calcium-dependent manner, yielding citrulline residues. Enzymatic citrullination abolishes positive charges of native protein molecules, inevitably causing significant alterations in their structure and function. Previously, we reported the abnormal accumulation of citrullinated proteins and an increase of PAD2 content in hippocampi of patients with Alzheimer disease. In this study, we investigated PAD expression by using dibutyryl cAMP (dbcAMP) in human astrocytoma U-251MG cells. Under normal culture conditions, PAD2 and PAD3 mRNA expression is detectable with quantitative PCR in U-251MG cells. The addition of dbcAMP in a dose-dependent manner significantly increased this mRNA expression and protein levels. Moreover, PAD enzyme activity also increased significantly and dose-dependently. Furthermore, the expression of PAD2 and PAD3 mRNA was inhibited by the cAMP-dependent PKA inhibitor KT5720, suggesting that such expression of dbcAMP-induced PAD2 and PAD3 mRNA is mediated by the cAMP-PKA signaling pathway in U-251MG cells. This is the first report to document the PAD2 and PAD3 mRNA expression induced by dbcAMP and to attribute the induction of these genes to mediation by the cAMP-PKA signaling pathway in U-251MG cells. © 2016 Wiley Periodicals, Inc.

Monomeric Form of Peptidylarginine Deiminase Type I Revealed by X-ray Crystallography and Small-Angle X-ray Scattering

Peptidylarginine deiminase (PAD; EC 3.5.3.15) is a post-translational modification enzyme that catalyzes the conversion of arginine in protein molecules to a citrulline residue in a Ca2+-dependent manner. In this study, we determined the structure of an active form of human PAD1 crystallized in the presence of Ca2+ at 3.2-Å resolution. Although human PAD2 and PAD4 isozymes were previously reported to form a head-to-tail homodimer, it is still unknown whether this quaternary structure is common to other PAD isozymes. The asymmetric unit of the crystal contained two PAD1 molecules; however, the head-to-tail dimeric form was not found. Small-angle X-ray scattering analyses revealed PAD1 to be a monomer in solution, while PAD3 was dimerized with a structure similar to PAD2 and PAD4. PAD1 was apparently different from the crystal structures of PAD2 and PAD4, with an elongated N-terminal loop that appears to prevent the formation of the homodimer. Of interest, the N-terminal loop occupied the substrate binding site of the adjacent PAD1 molecules in the crystal. Deimination of S100A3 peptides in vitro implied that PAD isozymes recognize the quaternary structure of S100A3. The substrate-accessible monomeric structure brought about by the extension of its N terminus may partly account for the highest tolerant substrate recognition of PAD1. This is the first ever report on the molecular structure of PAD1 demonstrating the unique monomeric form of the PAD isozyme.

Inherent ER stress in pancreatic islet β cells causes self-recognition by autoreactive T cells in type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease characterized by pancreatic β cell destruction induced by islet reactive T cells that have escaped central tolerance. Many physiological and environmental triggers associated with T1D result in β cell endoplasmic reticulum (ER) stress and dysfunction, increasing the potential for abnormal post-translational modification (PTM) of proteins. We hypothesized that β cell ER stress induced by environmental and physiological conditions generates abnormally-modified proteins for the T1D autoimmune response. To test this hypothesis we exposed the murine CD4+ diabetogenic BDC2.5 T cell clone to murine islets in which ER stress had been induced chemically (Thapsigargin). The BDC2.5 T cell IFNγ response to these cells was significantly increased compared to non-treated islets. This β cell ER stress increased activity of the calcium (Ca2+)-dependent PTM enzyme tissue transglutaminase 2 (Tgase2), which was necessary for full stress-dependent immunogenicity. Indeed, BDC2.5 T cells responded more strongly to their antigen after its modification by Tgase2. Finally, exposure of non-antigenic murine insulinomas to chemical ER stress in vitro or physiological ER stress in vivo caused increased ER stress and Tgase2 activity, culminating in higher BDC2.5 responses. Thus, β cell ER stress induced by chemical and physiological triggers leads to β cell immunogenicity through Ca2+-dependent PTM. These findings elucidate a mechanism of how β cell proteins are modified and become immunogenic, and reveal a novel opportunity for preventing β cell recognition by autoreactive T cells.

Functional characterization of T cells that recognize modified epitopes in patients with type 1 diabetes

Abstract Selective recognition of self-proteins that have undergone enzymatic post-translational modification (PTM) by CD4+ T cells is increasingly implicated as a means of potentiating the loss of tolerance in autoimmune diseases such as type 1 diabetes (T1D). In particular, enzymatic deamidation and citrullination of beta cell derived peptides at HLA anchor residues enhances their presentation by high risk alleles (DR4 and DQ8). Modification of residues outside of binding pockets alter T cell recognition, resulting in enhanced proliferation and increased cytokine production. Our work demonstrates that CD4+ T cells specific for PTM epitopes are present in the peripheral blood of patients with T1D at significantly higher ex vivo frequencies than in HLA matched controls. Upon re-activation, PTM specific T cells from T1D patients produce elevated levels of effector cytokines, most notably interferon gamma. Among T1D patients, we observed heterogeneous frequencies of PTM specific T cells with a range of functional profiles. This led us to hypothesize that activation of a PTM specific T cell response is of particular significance in a subset of T1D patients. To test this hypothesis we compared the ex vivo frequency and functional profiles of PTM specific T cells in a cross sectional cohort of patients selected to include diverse levels of residual c-peptide. Our findings implicate a mechanistic role for PTMs in exacerbating autoreactive T cell responses in T1D.

Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles

Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events.

Morroniside-Induced PP2A Activation Antagonizes Tau Hyperphosphorylation in a Cellular Model of Neurodegeneration.

An accumulation of hyperphosphorylated tau in the brain is a hallmark of Alzheimer's disease (AD). Deficits in protein phosphatase 2A (PP2A) are associated with tau hyperphosphorylation in AD. To investigate the effects of morroniside (MOR), isolated from Cornus officinalis, on tau hyperphosphorylation and its underlying mechanisms related to PP2A. SK-N-SH cells were pretreated with 50-200 μM MOR for 24 h followed by 20 nM okadaic acid (OA) for 6 h. PP2Ac siRNA was transfected into HEK293 cells to determine the direct interaction of MOR with PP2A. Western blotting was used to measure the expression of proteins and enzymes. PP2A activity was measured by molybdenum blue spectrophotometry. Pretreatment with MOR improved the cellular morphological damage and inhibited tau hyperphosphorylation in SK-N-SH cells induced by OA, a PP2A inhibitor. Moreover, MOR increased PP2A activity, concurrent with a decrease in the expression of demethylated PP2A at Leu309 and phosphorylated PP2A at Tyr307. MOR decreased protein phosphatase methylesterase 1 (PME-1) expression and the ratio of PME-1/leucine carboxyl methyltransferase 1 (LCMT-1). Furthermore, MOR treatment decreased the phosphorylation of Src at Tyr416, which regulates the phosphorylation of PP2A. MOR had no effect on PP2Ac expression and tau hyperphosphorylation in PP2Ac siRNA-transfected cells. MOR attenuated OA-induced tau hyperphosphorylation via PP2A activation, and its mechanism might be related to the regulation of PP2Ac post-translational modification and upstream enzymes such as Src and PME-1.

Stereospecific prenylation of tryptophan by a cyanobacterial post-translational modification enzyme

Prenylation is a key post-translational reaction to increase the structural diversity and bioactivity of peptides and proteins. Until now, only one post-translational modification enzyme, ComQ, has been identified to mediate the prenylation of a tryptophan residue in ribosomally synthesized peptides. Here, we report the in vitro characterization of KgpF, a novel prenyltransferase which transfers dimethylallyl moieties to tryptophan residues during kawaguchipeptin A biosynthesis. The stereospecific prenylation by KgpF was determined by a combination of in vitro dimethylallylation of Fmoc-tryptophan by KgpF and chemical synthesis of dimethylallylated Fmoc-tryptophan diastereomers. KgpF modified the tryptophan derivative with a dimethylallyl group at the 3 position of its indole ring, resulting in the formation of a tricyclic structure with the same scaffold as prenylation by ComQ, but with the opposite stereochemistry.

A pathway map of glutamate metabolism.

Glutamate metabolism plays a vital role in biosynthesis of nucleic acids and proteins. It is also associated with a number of different stress responses. Deficiency of enzymes involved in glutamate metabolism is associated with various disorders including gyrate atrophy, hyperammonemia, hemolytic anemia, γ-hydoxybutyric aciduria and 5-oxoprolinuria. Here, we present a pathway map of glutamate metabolism representing metabolic intermediates in the pathway, 107 regulator molecules, 9 interactors and 3 types of post-translational modifications. This pathway map provides detailed information about enzyme regulation, protein-enzyme interactions, post-translational modifications of enzymes and disorders due to enzyme deficiency. The information included in the map was based on published experimental evidence reported from mammalian systems.

Bioinformatics Knowledge Map for Analysis of Beta-Catenin Function in Cancer.

Given the wealth of bioinformatics resources and the growing complexity of biological information, it is valuable to integrate data from disparate sources to gain insight into the role of genes/proteins in health and disease. We have developed a bioinformatics framework that combines literature mining with information from biomedical ontologies and curated databases to create knowledge “maps” of genes/proteins of interest. We applied this approach to the study of beta-catenin, a cell adhesion molecule and transcriptional regulator implicated in cancer. The knowledge map includes post-translational modifications (PTMs), protein-protein interactions, disease-associated mutations, and transcription factors co-activated by beta-catenin and their targets and captures the major processes in which beta-catenin is known to participate. Using the map, we generated testable hypotheses about beta-catenin biology in normal and cancer cells. By focusing on proteins participating in multiple relation types, we identified proteins that may participate in feedback loops regulating beta-catenin transcriptional activity. By combining multiple network relations with PTM proteoform-specific functional information, we proposed a mechanism to explain the observation that the cyclin dependent kinase CDK5 positively regulates beta-catenin co-activator activity. Finally, by overlaying cancer-associated mutation data with sequence features, we observed mutation patterns in several beta-catenin PTM sites and PTM enzyme binding sites that varied by tissue type, suggesting multiple mechanisms by which beta-catenin mutations can contribute to cancer. The approach described, which captures rich information for molecular species from genes and proteins to PTM proteoforms, is extensible to other proteins and their involvement in disease.

Phosphorylation-dependent regulation of Notch1 signaling: the fulcrum of Notch1 signaling.

Notch signaling plays a pivotal role in cell fate determination, cellular development, cellular self-renewal, tumor progression, and has been linked to developmental disorders and carcinogenesis. Notch1 is activated through interactions with the ligands of neighboring cells, and acts as a transcriptional activator in the nucleus. The Notch1 intracellular domain (Notch1-IC) regulates the expression of target genes related to tumor development and progression. The Notch1 protein undergoes modification after translation by posttranslational modification enzymes. Phosphorylation modification is critical for enzymatic activation, complex formation, degradation, and subcellular localization. According to the nuclear cycle, Notch1-IC is degraded by E3 ligase, FBW7 in the nucleus via phosphorylation-dependent degradation. Here, we summarize the Notch signaling pathway, and resolve to understand the role of phosphorylation in the regulation of Notch signaling as well as to understand its relation to cancer. [BMB Reports 2015; 48(8): 431-437]

Mass spectrometric identification of citrullination sites and immunohistochemical detection of citrullinated glial fibrillary acidic protein in Alzheimer's disease brains.

Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that convert protein arginine to citrulline residues in a calcium ion-dependent manner. Previously, we reported the abnormal accumulation of citrullinated proteins and the increase in the amount of PAD2 in hippocampi from Alzheimer's disease (AD) patients. Moreover, glial fibrillary acidic protein (GFAP), an astrocyte-specific marker protein, and vimentin were identified as citrullinated proteins by using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. To clarify the substrate specificity of PADs against GFAP, we prepared recombinant human (rh)PAD1, rhPAD2, rhPAD3, rhPAD4, and rhGFAP. After incubation of rhGFAP with rhPAD1, rhPAD2, rhPAD3, and rhPAD4, citrullinated (cit-)rhGFAP was detected by Western blotting. The citrullination of rhGFAP by rhPAD2 was unique, specific, and time dependent; additionally, rhPAD1 slightly citrullinated rhGFAP. We then generated eight anti-cit-rhGFAP monoclonal antibodies, CTGF-125, -128, -129, -1212, -1213, -1221, -122R, and -1224R, which reacted specifically with cit-rhGFAP. Two of those eight monoclonal antibodies, CTGF-122R and -1224R, reacted with both cit-rhGFAP and rhGFAP in Western blots. By using the CTGF-1221 antibody and a tandem mass spectrometer, we identified the two independent citrullination sites (R270Cit and R416Cit) of cit-rhGFAP. Immunohistochemical analysis with CTGF-1221 antibody revealed cit-GFAP staining in the hippocampus of AD brain, and the cit-GFAP-positive cells appeared to be astrocyte-like cells. These collective results strongly suggest that PAD2 is responsible for the citrullination of GFAP in the progression of AD and that the monoclonal antibody CTGF-1221, reacting with cit-GFAP at R270Cit and R416Cit, is useful for immunohistochemical investigation of AD brains.

Modularity of RiPP Enzymes Enables Designed Synthesis of Decorated Peptides

Macrocyclases and other posttranslational enzymes afford derived peptides with improved properties for pharmaceutical and biotechnological applications. Here, we asked whether multiple posttranslational modifications could be simultaneously controlled and matched to rationally generate new peptide derivatives. We reconstituted the cyanobactin peptide natural products in vitro with up to five different posttranslational enzymes in a single tube. By manipulating the order of addition and identity of enzymes and exploiting their broad-substrate tolerance, we engineered the production of highly unnatural derivatives, including an N-C peptide macrocycle of 22 amino acids in length. In addition to engineering, this work better defines the macrocyclization mechanism, provides the first biochemical demonstration of Ser/Thr posttranslational prenylation, and is the first example of reconstitution of a native, multistep RiPP pathway with multiple enzymes in one pot. Overall, this work demonstrates how the modularity of posttranslational modification enzymes can be used to design and synthesize desirable peptide motifs.

Molecular Diversity and Gene Evolution of the Venom Arsenal of Terebridae Predatory Marine Snails.

Venom peptides from predatory organisms are a resource for investigating evolutionary processes such as adaptive radiation or diversification, and exemplify promising targets for biomedical drug development. Terebridae are an understudied lineage of conoidean snails, which also includes cone snails and turrids. Characterization of cone snail venom peptides, conotoxins, has revealed a cocktail of bioactive compounds used to investigate physiological cellular function, predator-prey interactions, and to develop novel therapeutics. However, venom diversity of other conoidean snails remains poorly understood. The present research applies a venomics approach to characterize novel terebrid venom peptides, teretoxins, from the venom gland transcriptomes of Triplostephanus anilis and Terebra subulata. Next-generation sequencing and de novo assembly identified 139 putative teretoxins that were analyzed for the presence of canonical peptide features as identified in conotoxins. To meet the challenges of de novo assembly, multiple approaches for cross validation of findings were performed to achieve reliable assemblies of venom duct transcriptomes and to obtain a robust portrait of Terebridae venom. Phylogenetic methodology was used to identify 14 teretoxin gene superfamilies for the first time, 13 of which are unique to the Terebridae. Additionally, basic local algorithm search tool homology-based searches to venom-related genes and posttranslational modification enzymes identified a convergence of certain venom proteins, such as actinoporin, commonly found in venoms. This research provides novel insights into venom evolution and recruitment in Conoidean predatory marine snails and identifies a plethora of terebrid venom peptides that can be used to investigate fundamental questions pertaining to gene evolution.