Kevin L. Bicker

Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation

PAD4 has been strongly implicated in the pathogenesis of autoimmune, cardiovascular and oncological diseases, through clinical genetics and gene disruption in mice. Novel, selective PAD4 inhibitors binding to a calcium-deficient form of the PAD4 enzyme have, for the first time, validated the critical enzymatic role of human and mouse PAD4 in both histone citrullination and neutrophil extracellular trap formation. The therapeutic potential of PAD4 inhibitors can now be explored.

Peptidylarginine deiminase 2-catalyzed histone H3 arginine 26 citrullination facilitates estrogen receptor α target gene activation

Cofactors for estrogen receptor α (ERα) can modulate gene activity by posttranslationally modifying histone tails at target promoters. Here, we found that stimulation of ERα-positive cells with 17β-estradiol (E2) promotes global citrullination of histone H3 arginine 26 (H3R26) on chromatin. Additionally, we found that the H3 citrulline 26 (H3Cit26) modification colocalizes with ERα at decondensed chromatin loci surrounding the estrogen-response elements of target promoters. Surprisingly, we also found that citrullination of H3R26 is catalyzed by peptidylarginine deiminase (PAD) 2 and not by PAD4 (which citrullinates H4R3). Further, we showed that PAD2 interacts with ERα after E2 stimulation and that inhibition of either PAD2 or ERα strongly suppresses E2-induced H3R26 citrullination and ERα recruitment at target gene promoters. Collectively, our data suggest that E2 stimulation induces the recruitment of PAD2 to target promoters by ERα, whereby PAD2 then citrullinates H3R26, which leads to local chromatin decondensation and transcriptional activation.

The protein arginine deiminases: Structure, function, inhibition, and disease†

The post-translational modification of histones has significant effects on overall chromatin function. One such modification is citrullination, which is catalyzed by the protein arginine deiminases (PADs), a unique family of enzymes that catalyzes the hydrolysis of peptidyl-arginine to form peptidyl-citrulline on histones, fibrinogen, and other biologically relevant proteins. Overexpression and/or increased PAD activity is observed in several diseases, including rheumatoid arthritis, Alzheimers disease, multiple sclerosis, lupus, Parkinsons disease, and cancer. This review discusses the important structural and mechanistic characteristics of the PADs, as well as recent investigations into the role of the PADs in increasing disease severity in RA and colitis and the importance of PAD activity in mediating neutrophil extracellular trap formation through chromatin decondensation. Lastly, efforts to develop PAD inhibitors with excellent potency, selectivity and in vivo efficacy are discussed, highlighting the most promising inhibitors.

Peptide Borono Lectins (PBLs): A New Tool for Glycomics and Cancer Diagnostics

A growing body of evidence supports the idea that during tumorgenesis aberrant glycosylation events occur to cell surface and secreted glycoproteins as well as glycolipids, and as a result the glycans produced by cancer cells differ in both structure and level to those produced by normal cells. 2] These changes generally arise from the altered expression of glycosyl-transferases; typically leading to increased branching in core glycan structures as well as altering the terminal saccharide structures. The appearance of a variety of sialylated and fucosylated terminal glycan structures [for example, sialyl Lewis X (sLe), sialyl Lewis A (sLe), sialyl Tn (sTn), and Lewis Y (Le)] has been associated with malignancy. Whereas it is unclear whether these changes in glycan content are a cause or effect of oncogenesis, it is clear that specific cell surface glycans can contribute to the metastatic potential of particular tumor types. Regardless of their specific role in oncogenesis, the expression of these various glycan structures is dependent on both the tumor type and the stage of the disease; thus their ACHTUNGTRENNUNGappearance can be exploited for the development of novel cancer diagnostics. In fact, some tests based on aberrant glycosylation events have been developed for initial detection and for monitoring disease progression after beginning therapy. For example, an immunoassay for carcinoembryonic antigen (CEA), an aberrantly glycosylated glycoprotein, measures serum levels of CEA to monitor disease progression in colon cancer patients after surgery. Whereas these tests are of great value, they are often expensive and time consuming and cheaper more efficient analyses are needed. Boronic acids and other small molecules have shown great utility in sensing simple sugars and complex glycoproteins. It is therefore reasonable that the differential or random display of phenylboronic acid (PBA) moieties on a peptide backbone would result in a biocompatible, water soluble, cancer diagnostic (Figure 1A), overcoming the limitations of other boronic acid based sensors. Herein, we report proof of concept for: 1) the design and synthesis of a peptide borono lectin (PBL) ACHTUNGTRENNUNGlibrary; 2) that selective and cross-reactive PBLs can be identified using conventional microtiter plate screening techniques; and 3) that these compounds show utility for binding cancer related targets. A “low” diversity 12-mer PBL library was synthesized on aminomethyl PEG–PS resin (100 mm) using a biased split-and-pool combinatorial approach (Figure 1B). This resin was chosen because it is stable to acid hydrolysis, displays limited inherent fluorescence, and facilitates ligand-binding interactions comparable to that observed in solution. Standard Fmoc synthetic schemes were followed with Dde protected side chains. Ten amino acids were randomized between alanine endcaps producing a library with a general sequence of: Cbz-A-(X)10-A-resin where X is either 2,3-diaminopropanoic acid (DPR), 2,4-diaminobutanoic acid (DAB), ornithine (Orn), lysine (Lys), or alanine (Ala). The diamino acids chosen contain one, two, three, or four methylene units, respectively, spacing the side-chain amine from the peptide backbone. This “vertical” positioning alters the orientation between PBAs depending on the number of -CH2groups in the side chains whereas variation in the peptide sequence modifies the “horizontal” arrangement. Distinct positional variation was thereby generated between PBAs within a sequence, creating unique geometric constraints for binding. Introduction of PBAs was accomplished by removing the Dde protecting groups with hydrazine, coupling the side-chain amine with excess 2-formylphenylboronic acid, followed by reduction with NaBH4. This route to install the PBA benefits from intramolecular Lewis acid catalysis, provided by the boronic acid, to insure near quantitative incorporation of the desired functionality (see the Supporting Information for details). The theoretical diversity of the resulting PBL library (5) is in the order of 10 million distinct peptide sequences containing a statistical average of 4 PBA moieties per peptide. Because 1 g Figure 1. A) Schematic representation of a phenylboronic acid substituted peptide (PBL, sequence chosen at random) binding to a glycan or glycoprotein. B) Biased split-and-pool method used to generate the “low” diversity PBL library.

Seeing citrulline: development of a phenylglyoxal-based probe to visualize protein citrullination.

Protein arginine deiminases (PADs) catalyze the hydrolysis of peptidyl arginine to form peptidyl citrulline. Abnormally high PAD activity is observed in a host of human diseases, but the exact role of protein citrullination in these diseases and the identities of specific citrullinated disease biomarkers remain unknown, largely because of the lack of readily available chemical probes to detect protein citrullination. For this reason, we developed a citrulline-specific chemical probe, rhodamine-phenylglyoxal (Rh-PG), which we show can be used to investigate protein citrullination. This methodology is superior to existing techniques because it possesses higher throughput and excellent sensitivity. Additionally, we demonstrate that this probe can be used to determine the kinetic parameters for a number of protein substrates, monitor drug efficacy, and identify disease biomarkers in an animal model of ulcerative colitis that displays aberrantly increased PAD activity.

Boronic acid functionalized peptidyl synthetic lectins: combinatorial library design, peptide sequencing, and selective glycoprotein recognition

Aberrant glycosylation of cell membrane and secreted glycoproteins is a hallmark of various disease states, including cancer. The natural lectins currently used in the recognition of these glycoproteins are costly, difficult to produce, and unstable toward rigorous use. Herein we describe the design and synthesis of several boronic acid functionalized peptide-based synthetic lectin (SL) libraries, as well as the optimized methodology for obtaining peptide sequences of these SLs. SL libraries were subsequently used to identify SLs with as high as 5-fold selectivity for various glycoproteins. SLs will inevitably find a role in cancer diagnostics, given that they do not suffer from the drawbacks of natural lectins and that the combinatorial nature of these libraries allows for the identification of an SL for nearly any glycosylated biomolecule.

Synovial fibroblast-neutrophil interactions promote pathogenic adaptive immunity in rheumatoid arthritis

Neutrophil extracellular traps containing citrullinated peptides promote inflammation in synovial fibroblasts. NETs Blow the Joint Neutrophil extracellular traps (NETs) activate synovial fibroblast-like synoviocytes (FLS) in joints of individuals with rheumatoid arthritis. Now, Carmona-Rivera et al. investigate the mechanism behind this activation. They found that NETs containing citrullinated peptides could be internalized by FLS through the RAGE-TLR9 pathway and then loaded onto major histocompatibility complex class II and presented to antigen-specific T cells, which contribute to joint inflammation. NET-loaded FLS induced autoantibody production and joint disease in mice. These data suggest that cross-talk between NETs and FLS may contribute to rheumatoid arthritis. Rheumatoid arthritis (RA) is characterized by synovial joint inflammation and by development of pathogenic humoral and cellular autoimmunity to citrullinated proteins. Neutrophil extracellular traps (NETs) are a source of citrullinated autoantigens and activate RA synovial fibroblast-like synoviocytes (FLS), cells crucial in joint damage. We investigated the molecular mechanisms by which NETs promote proinflammatory phenotypes in FLS and whether these interactions generate pathogenic anti-citrulline adaptive immune responses. NETs containing citrullinated peptides are internalized by FLS through a RAGE-TLR9 pathway, promoting FLS inflammatory phenotype and their up-regulation of major histocompatibility complex (MHC) class II. Once internalized, arthritogenic NET peptides are loaded into FLS MHC class II and presented to antigen-specific T cells. HLA-DRB1*04:01 transgenic mice immunized with mouse FLS loaded with NETs develop antibodies specific to citrullinated forms of relevant autoantigens implicated in RA pathogenesis as well as cartilage damage. These results implicate FLS as notable mediators in RA pathogenesis, through the internalization and presentation of NET citrullinated peptides to the adaptive immune system, leading to pathogenic autoimmunity and cartilage damage.

Synthetic lectin arrays for the detection and discrimination of cancer associated glycans and cell lines

Aberrant glycosylation is a hallmark of various disease states, including cancer, and effective detection and discrimination between healthy and diseased cells is an important challenge for the diagnosis and treatment of many diseases. Here, we describe the use of boronic acid functionalized synthetic lectins (SLs) in an array format for the differentiation of structurally similar cancer associated glycans and cancer cell lines; discrimination is based on subtle variations in glycosylation patterns. We further demonstrate the utility of our SLs in recognizing glycoproteins with up to 50-fold selectivity, even in 95% human serum. Given their robust and selective nature, these SLs were able to effectively distinguish (a) five structurally similar glycans with 94% accuracy; (b) seven normal, cancerous and metastatic colon cancer cell lines, including three isogenic cell lines, with 92% accuracy; and (c) these same seven cell lines using a guided statistical analysis to improve our analysis to 97% accuracy. In total, these data suggest that an SL-based array will be useful for the diagnosis of cancer.

A combinatorial approach to characterize the substrate specificity of protein arginine methyltransferase 1.

The dysregulation of protein arginine methyltransferases (PRMTs) is implicated in a wide variety of disease states. Here we report the design, synthesis, and screening of a combinatorial peptide library used to characterize the substrate specificity of PRMT1. The information gained from this approach was used to develop a PRMT1 inhibitor with enhanced selectivity.

Chemical Proteomic Platform To Identify Citrullinated Proteins

Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and are routinely used for disease diagnosis. Protein citrullination is also increased in cancer and other autoimmune disorders, suggesting that citrullinated proteins may serve as biomarkers for diseases beyond RA. To identify these citrullinated proteins, we developed biotin-conjugated phenylglyoxal (biotin-PG). Using this probe and our platform technology, we identified >50 intracellular citrullinated proteins. More than 20 of these are involved in RNA splicing, suggesting, for the first time, that citrullination modulates RNA biology. Overall, this chemical proteomic platform will play a key role in furthering our understanding of protein citrullination in rheumatoid arthritis and potentially a wider spectrum of inflammatory diseases.