An improved synthesis of haloacetamidine-based inactivators of protein arginine deiminase 4 (PAD4)

Protein arginine deiminase 4 (PAD4) is an enzyme that hydrolyzes peptidyl arginine residues to form citrulline and ammonia. This enzyme has been implicated in several disease states, for example, rheumatoid arthritis, and therefore represents a unique target for the development of a novel therapeutic. A solution-phase synthesis of Cl-amidine, the most potent PAD4 inactivator described to date, has been developed. This synthesis proceeds in 80% yield over four steps at a significantly (12-fold) lower cost.     

Singlet oxygen generation versus O–O homolysis in phenyl-substituted anthracene endoperoxides investigated by RASPT2, CASPT2, CC2, and TD-DFT methods

Peptidylarginine deiminase 4 (PAD4), also known as protein arginine deiminase 4, performs a post-translational deimination that converts arginine to citrulline. The dysregulation of PAD4 has been implicated in a number of diseases, including rheumatoid arthritis (RA) and cancer. This makes PAD4 an important therapeutic target. To develop small-molecule inhibitors as potential treatments, it is advantageous if the catalytic mechanism is well understood. The protonation states of the active site residues, which have long been under controversy, have a direct impact on the catalytic mechanism. Two competing mechanisms are under investigation in the current literature. The first is a reverse protonation mechanism that depends on the active site histidine and cysteine existing as an ion pair. The second is a substrate-assisted mechanism that depends on the active site histidine and cysteine being neutral. This study uses the semimicroscopic protein dipoles Langevin dipoles (PDLD/S) linear response approximation method in the MOLARIS software package to calculate the change in solvation energy of moving the residue from water to the protein interior, and then using that information to assess the protonation states of the active site residues of PAD4. Results from these calculations suggest that in the enzyme–substrate complex of PAD4, the cysteine and histidine are protonated and deprotonated, respectively, and are therefore both neutral, analogous to the proposed protonation states of the active site residues in the Michaelis complex in the substrate-assisted mechanism.     

Discovery of new inhibitor for the protein arginine deiminase type 4 (PAD4) by rational design of α-enolase-derived peptides

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting about 0.24 % of the world population. Protein arginine deiminase type 4 (PAD4) is believed to be responsible for the occurrence of RA by catalyzing citrullination of proteins. The citrullinated proteins act as autoantigens by stimulating an immune response. Citrullinated α-enolase has been identified as one of the autoantigens for RA. Hence, α-enolase serves as a suitable template for design of potential peptide inhibitors against PAD4. The binding affinity of α-enolase-derived peptides and PAD4 was virtually determined using PatchDock and HADDOCK docking programs. Synthesis of the designed peptides was performed using a solid phase peptide synthesis method. The inhibitory potential of each peptide was determined experimentally by PAD4 inhibition assay and IC₅₀ measurement. PAD4 assay data show that the N-P2 peptide is the most favourable substrate among all peptides. Further modification of N-P2 by changing the Arg residue to canavanine [P2 (Cav)] rendered it an inhibitor against PAD4 by reducing the PAD4 activity to 35 % with IC₅₀ 1.39 mM. We conclude that P2 (Cav) is a potential inhibitor against PAD4 and can serve as a starting point for the development of even more potent inhibitors.     

An in silico analysis of primary and secondary structure specificity determinants for human peptidylarginine deiminase types 2 and 4

Human peptidylarginine deiminases (hPADs) are a family of five calcium-dependent enzymes that facilitate citrullination, which is the post-translational modification of peptidyl arginine to peptidyl citrulline. The isozymes hPAD2 and hPAD4 have been implicated in the development and progression of several autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. To better characterize the primary and secondary structure determinants of citrullination specificity, we mined the literature for protein sequences susceptible to citrullination by hPAD2 or hPAD4. First, protein secondary structure classification (α-helix, β-sheet, or coil) was predicted using the PSIPRED software. Next, we used motif-x and pLogo to extract and visualize statistically significant motifs within each data set. Within the data sets of peptides predicted to lie in coil regions, both hPAD2 and hPAD4 appear to favor citrullination of glycine-containing motifs, while distinct hydrophobic motifs were identified for hPAD2 citrullination sites predicted to reside within α-helical and β-sheet regions. Additionally, we identified potential substrate overlap between coil region citrullination and arginine methylation. Together, these results confirm the importance and offer some insight into the role of secondary structure elements for citrullination specificity, and provide biological context for the existing hPAD specificity and arginine post-translational modification literature.     

Halogen Bonding Increases the Potency and Isozyme Selectivity of Protein Arginine Deiminase 1 Inhibitors

Protein arginine deiminases (PADs) hydrolyze the side chain of arginine to form citrulline. Aberrant PAD activity is associated with rheumatoid arthritis, multiple sclerosis, lupus, and certain cancers. These pathologies established the PADs as therapeutic targets and multiple PAD inhibitors are known. Herein, we describe the first highly potent PAD1‐selective inhibitors ( 1 and 19 ). Detailed structure–activity relationships indicate that their potency and selectivity is due to the formation of a halogen bond with PAD1. Importantly, these inhibitors inhibit histone H3 citrullination in HEK293TPAD1 cells and mouse zygotes with excellent potency. Based on this scaffold, we also developed a PAD1‐selective activity‐based probe that shows remarkable cellular efficacy and proteome selectivity. Based on their potency and selectivity we expect that 1 and 19 will be widely used chemical tools to understand PAD1 biology.     

Arginine Deiminase Induces Immunogenic Cell Death and Is Enhanced by N-acetylcysteine in Murine MC38 Colorectal Cancer Cells and MDA-MB-231 Human Breast Cancer Cells In Vitro

The use of arginine deiminase (ADI) for arginine depletion therapy is an attractive anticancer approach. Combination strategies are needed to overcome the resistance of severe types of cancer cells to this monotherapy. In the current study, we report, for the first time, that the antioxidant N-acetylcysteine (NAC), which has been used in therapeutic practices for several decades, is a potent enhancer for targeted therapy that utilizes arginine deiminase. We demonstrated that pegylated arginine deiminase (ADI-PEG 20) induces apoptosis and G0/G1 phase arrest in murine MC38 colorectal cancer cells; ADI-PEG 20 induces Ca²⁺ overload and decreases the mitochondrial membrane potential in MC38 cells. ADI-PEG 20 induced the most important immunogenic cell death (ICD)-associated feature: cell surface exposure of calreticulin (CRT). The antioxidant NAC enhanced the antitumor activity of ADI-PEG 20 and strengthened its ICD-associated features including the secretion of high mobility group box 1 (HMGB1) and adenosine triphosphate (ATP). In addition, these regimens resulted in phagocytosis of treated MC38 cancer cells by bone marrow-derived dendritic cells (BMDCs). In conclusion, we describe, for the first time, that NAC in combination with ADI-PEG 20 not only possesses unique cytotoxic anticancer properties but also triggers the hallmarks of immunogenic cell death. Hence, ADI-PEG 20 in combination with NAC may represent a promising approach to treat ADI-sensitive tumors while preventing relapse and metastasis.     

Enzymatic Modification of Soluble Cyanophycin Using the Type II Peptidyl Arginine Deiminase from Oryctolagus cuniculus

An increased structural variety expands the number of putative applications for cyanophycin (multi‐l ‐arginyl‐poly‐[l ‐aspartic acid], CGP). Therefore, structural modifications of CGP are of major interest; these are commonly obtained by modification and optimization of the bacterial producing strain or by chemical modification. In this study, an enzymatic modification of arginine side chains from lysine‐rich CGP is demonstrated using the peptidyl arginine deiminase from Oryctolagus cuniculus, purified from Escherichia coli after heterologous expression. About 10% of the arginine side chains are converted to citrulline which corresponds to 4% of the polymer's total side chains. An inhibition of the reaction in the presence of small amounts of l ‐citrulline is observed, thereby explaining the low conversion rate. CGP dipeptides can be modified with about 7.5 mol% of the Asp‐Arg dipeptides being converted to Asp‐Cit. These results show that the enzymatic modification of CGP is feasible, opening up a whole new area of possible CGP modifications for further research.

     

Arginine deiminase uses an active-site cysteine in nucleophilic catalysis of L-arginine hydrolysis

Arginine deiminase (EC 3.5.3.6) catalyzes the hydrolysis of l-arginine to citrulline and ammonium ion, which is the first step of the microbial l-arginine degradation pathway. The deiminase conserves the active-site Cys-His-Asp motif found in several related enzymes that catalyze group-transfer reactions from the guanidinium center of arginine-containing substrates. For each of these enzymes, nucleophilic catalysis by the conserved Cys has been postulated but never tested. In this communication we report the results from rapid quench studies of single-turnover reactions carried out with recombinant Pseudomonas aeruginosa arginine deiminase and limiting [14C-1]l-arginine. The citrulline-formation and arginine-decay curves measured at 25 degrees C were fitted to yield apparent rate constants k = 3.6 +/- 0.1 s-1 and k = 4.2 +/- 0.1 s-1, respectively. The time course for the formation (k =13 s-1) and decay (k = 6.5 s-1) of 14C-labeled enzyme defined a kinetically competent intermediate. Under the same reaction conditions, the Cys406Ser mutant failed to form the 14C-labeled enzyme intermediate. These results, along with the recently reported enzyme X-ray structure (Galkin, A.; Kulakova, L.; Sarikaya, E.; Lim, K.; Howard, A.; Herzberg, O. J. Biol. Chem. 2004, 279, 14001-14008, evidence a reaction pathway in which l-arginine deimination proceeds via a covalent enzyme intermediate formed by ammonia displacement from the arginine guanidinum carbon by the active-site Cys406.     

Self-reporting fluorescent substrates of protein tyrosine kinases

A new mechanistic principle by which protein tyrosine kinase substrates fluorescently report the introduction of a phosphate moiety has been developed. NMR was used to establish that tyrosine phosphorylation induces the disruption of pi-pi stacking interactions of the tyrosine moiety with a proximal fluorophore on the peptide substrate. We have demonstrated that (1) the peptide substrates described in this study are useful for a wide variety of different tyrosine kinases, (2) physiological concentrations of ATP can be employed (unlike the standard radioactive ATP kinase assays), thus providing a more realistic assessment of inhibitor potency, and (3) protein kinase self-activation can be observed in real-time.     

Molecular dynamics and density functional studies of substrate binding and catalysis of arginine deiminase

The active-site dynamics of arginine deiminase (ADI) complexed with the arginine substrate are investigated with ns molecular dynamics for the wildtype ADI and several mutants. It is shown that the substrate is held in the active site by an extensive hydrogen bond network, which may be weakened by substitution of active-site residues. In addition, the initial step of the catalysis is explored in several truncated active-site models with density functional theory. Evidence is presented in support of the hypothesis that the nucleophilic attack of the ADI Cys thiol at the guanidino carbon of the substrate is initiated by substrate-mediated proton transfer to a His residue in the catalytic triad (Cys-His-Glu). In addition, the active-site residues are found to strongly influence the reaction profile, consistent with their important role in catalysis.     

L-canavanine is a time-controlled mechanism-based inhibitor of Pseudomonas aeruginosa arginine deiminase

The mechanism for inhibition of the Pseudomonas aeruginosa arginine deiminase (PaADI) by the arginine analogue l-canavanine was investigated. Inhibition by this substance (kinact = 0.31 +/- 0.03 min-1 and Ki = 1.7 +/- 0.5 mM) is associated with the formation of a modestly stable S-alkylthiouronium intermediate, detected by using kinetic techniques and identified by using electrospray ionization mass spectrometry. The electronic and/or orientation effects, caused by oxygen-for-methylene substitution in l-canavanine, on the rate of enzyme regeneration from the S-alkylthiouronium intermediate could serve as the basis for a strategy for the rational design of new slow substrate inhibitors of ADI.     

Detection of Mycoplasma Contamination in Lymphoblastoid Cultures by a Simple,HPLC Method

Abstract

A simple method for detecting mycoplasma contamination of lymphoid cell cultures is discussed. This method is based on high pressure liquid chromatographic (HPLC) detection of citrulline by reversed-phase separation of its ortho-phthaldialdehyde (OPA) derivative. Formation of citrulline via conversion of arginine using the enzyme arginine deiminase present in most of the commonly encountered strains of mycoplasma is measured by this sensitive technique. The use of HPLC techniques offers an extremely sensitive indication of mycoplasma contamination in cells having large nuclei which make detection by staining with Hoechst dyes difficult to interpret.     

化学-生物法制备D-精氨酸和L-瓜氨酸

在水溶液中以水杨醛为催化剂,L-精氨酸可以快速消旋为DL-精氨酸．后以DL-精氨酸为底物,利用粪链球菌（Streptococcus faecalis）精氨酸脱亚胺酶对L-精氨酸的专一脱亚胺作用,同时制备D精氨酸和L-瓜氨酸,分别以92．3％,94．2％的产率获得光学纯的D-精氨酸和L-瓜氨酸．     

Fast and efficient MCR-based synthesis of clickable rhodamine tags for protein profiling

Protein profiling probes are important tools for studying the composition of the proteome and as such have contributed greatly to the understanding of various complex biological processes in higher organisms. For this purpose the application of fluorescently labeled activity or affinity probes is highly desirable. Especially for in vivo detection of low abundant target proteins, otherwise difficult to analyse by standard blotting techniques, fluorescently labeled profiling probes are of high value. Here, a one-pot protocol for the synthesis of activated fluorescent labels (i.e. azide, alkynyl or NHS), based on the Ugi-4-component reaction (Ugi-4CR), is presented. As a result of the peptoidic structure formed, the fluorescent properties of the products are pH insensitive. Moreover, the applicability of these probes, as exemplified by the labeling of model protein BSA, will be discussed.     

Detection of creatinine using surface-driven ordering transitions of liquid crystals

Determining creatinine levels in blood is of great importance in the detection of high risk for renal failure. Here, we report a simple methodology for real-time monitoring of creatinine employing surface-driven ordering transitions in liquid crystals (LCs) by changing pH in presence of creatinine deiminase enzyme. It is found that when 5CB (4-Cyano-4?-pentylbiphenyl) LC doped with 4?-hexyl-biphenyl-4-carboxylic acid, a bright optical appearance was observed (at aqueous–LC interface) which is not disturbed in presence of creatinine, consistent with a planar/tilted orientation of the LC molecules at those interface. Interestingly, in presence of creatinine deiminase, an ordering transition was observed resulting from enzymatic reactions (giving rise to NH₄⁺ ions) that can change the local pH values and lead to dark optical appearance of the LC. Presence of different amounts of creatinine would lead varied ordering transition that can be monitored in real time in presence of creatinine deiminase. Our approach could detect the creatinine levels as low as that of the healthy adult (~50 µM) and can be successfully applied to measure higher concentration of creatinine in real time using dynamic optical response of the LC.     

Determination of sites citrullinated by peptidylarginine deiminase using 18O stable isotope labeling and mass spectrometry

Peptidylarginine deiminase (PADI) is an enzyme which catalyzes conversion of arginine residues into citrulline residues in proteins. Citrullination is known to be related to autoimmune diseases including rheumatoid arthritis. Previous work in this laboratory succeeded in identifying citrullinated sites of human fibrinogen by mass spectrometry, but discrimination between citrullination and deamidation of asparagines and glutamine required time-consuming and labor-intensive inspection of tandem mass spectra. In this work a stable isotope is utilized to improve on a previous method for the determination of citrullinated sites by mass spectrometry. Since an oxygen atom is incorporated into the citrulline residue from H(2)O in citrullination by PADI, peptides citrullinated in 50% H(2)(18)O would show a characteristic isotope distribution different from natural abundance, and thus determination of citrullinated sites is expected to be much easier. To verify the utility of this new method, the sites of citrullination of human fibrinogen by human PADI4 were investigated using 50% H(2)(18)O. Compared with the previous method, this new method identified citrullinated sites more easily and effectively, while both the determined citrullinated sites and protein sequence coverage were unaltered.     

Characterizing Enzyme Cooperativity with Imaging SAMDI-MS

This paper describes a method that combines a microfluidic device and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI) mass spectrometry to calculate the cooperativity in binding of calcium ions to peptidylarginine deiminase type 2 (PAD2). This example uses only 120 μL of enzyme solution and three fluidic inputs. This microfluidic device incorporates a self-assembled monolayer that is functionalized with a peptide substrate for PAD2. The enzyme and different concentrations of calcium ions are flowed through each of eight channels, where the position along the channel corresponds to reaction time and position across the channel corresponds to the concentration of Ca²⁺. Imaging SAMDI (iSAMDI) is then used to determine the yield for the enzyme reaction at each 200 μm pixel on the monolayer, providing a time course for the reactions. Analysis of the peptide conversion as a function of position and time gives the degree of cooperativity (n) and the concentration of ligand required for half maximal activity (K_0.5) for the Ca²⁺ – dependent activation of PAD2. This work establishes a high-throughput and label-free method for studying enzyme-ligand binding interactions and widens the applicability of microfluidics and matrix-assisted laser desorption/ionization mass spectrometry (MALDI) imaging mass spectrometry.