Citrullination-Resistant LL-37 Is a Potent Antimicrobial Agent in the Inflammatory Environment High in Arginine Deiminase Activity.

LL-37, the only member of the mammalian cathelicidin in humans, plays an essential role in innate immunity by killing pathogens and regulating the inflammatory response. However, at an inflammatory focus, arginine residues in LL-37 can be converted to citrulline via a reaction catalyzed by peptidyl-arginine deiminases (PAD2 and PAD4), which are expressed in neutrophils and are highly active during the formation of neutrophil extracellular traps (NETs). Citrullination impairs the bactericidal activity of LL-37 and abrogates its immunomodulatory functions. Therefore, we hypothesized that citrullination-resistant LL-37 variants would retain the functionality of the native peptide in the presence of PADs. To test this hypothesis, we synthetized LL-37 in which arginine residues were substituted by homoarginine (hArg-LL-37). Bactericidal activity of hArg-LL-37 was comparable with that of native LL-37, but neither treatment with PAD4 nor exposure to NETs affected the antibacterial and immunomodulatory activities of hArg-LL-37. Importantly, the susceptibilities of LL-37 and hArg-LL-37 to degradation by proteases did not significantly differ. Collectively, we demonstrated that citrullination-resistant hArg-LL-37 is an attractive lead compound for the generation of new agents to treat bacterial infections and other inflammatory diseases associated with enhanced PAD activity. Moreover, our results provide a proof-of-concept for synthesis of therapeutic peptides using homoarginine.

Carbon Nanoparticles Inhibit the Antimicrobial Activities of the Human Cathelicidin LL-37 through Structural Alteration.

Host defense peptides, also known as antimicrobial peptides, are key elements of innate host defense. One host defense peptide with well-characterized antimicrobial activity is the human cathelicidin, LL-37. LL-37 has been shown to be upregulated at sites of infection and inflammation and is regarded as one of the primary innate defense molecules against bacterial and viral infection. Human exposure to combustion-derived or engineered nanoparticles is of increasing concern, and the implications of nanomaterial exposure on the human immune response is poorly understood. However, it is widely acknowledged that nanoparticles can interact strongly with several immune proteins of biological significance, with these interactions resulting in structural and functional changes of the proteins involved. This study investigated whether the potent antibacterial and antiviral functions of LL-37 were inhibited by exposure to, and interaction with, carbon nanoparticles, together with characterizing the nature of the interaction. LL-37 was exposed to carbon black nanoparticles in vitro, and the antibacterial and antiviral functions of the peptide were subsequently assessed. We demonstrate a substantial loss of antimicrobial function when the peptide was exposed to low concentrations of nanomaterials, and we further show that the nanomaterial-peptide interaction resulted in a significant change in the structure of the peptide. The human health implications of these findings are significant, as, to our knowledge, this is the first evidence that nanoparticles can alter host defense peptide structure and function, indicating a new role for nanoparticle exposure in increased disease susceptibility.

Monothiol glutaredoxins can bind linear [Fe3S4]+ and [Fe4S4]2+ clusters in addition to [Fe2S2]2+ clusters: spectroscopic characterization and functional implications.

Saccharomyces cerevisiae mitochondrial glutaredoxin 5 (Grx5) is the archetypical member of a ubiquitous class of monothiol glutaredoxins with a strictly conserved CGFS active-site sequence that has been shown to function in biological [Fe2S2](2+) cluster trafficking. In this work, we show that recombinant S. cerevisiae Grx5 purified aerobically, after prolonged exposure of the cell-free extract to air or after anaerobic reconstitution in the presence of glutathione, predominantly contains a linear [Fe3S4](+) cluster. The excited-state electronic properties and ground-state electronic and vibrational properties of the linear [Fe3S4](+) cluster have been characterized using UV-vis absorption/CD/MCD, EPR, Mössbauer, and resonance Raman spectroscopies. The results reveal a rhombic S = 5/2 linear [Fe3S4](+) cluster with properties similar to those reported for synthetic linear [Fe3S4](+) clusters and the linear [Fe3S4](+) clusters in purple aconitase. Moreover, the results indicate that the Fe-S cluster content previously reported for many monothiol Grxs has been misinterpreted exclusively in terms of [Fe2S2](2+) clusters, rather than linear [Fe3S4](+) clusters or mixtures of linear [Fe3S4](+) and [Fe2S2](2+) clusters. In the absence of GSH, anaerobic reconstitution of Grx5 yields a dimeric form containing one [Fe4S4](2+) cluster that is competent for in vitro activation of apo-aconitase, via intact cluster transfer. The ligation of the linear [Fe3S4](+) and [Fe4S4](2+) clusters in Grx5 has been assessed by spectroscopic, mutational, and analytical studies. Potential roles for monothiol Grx5 in scavenging and recycling linear [Fe3S4](+) clusters released during protein unfolding under oxidative stress conditions and in maturation of [Fe4S4](2+) cluster-containing proteins are discussed in light of these results.

Monothiol glutaredoxins function in storing and transporting [Fe2S2] clusters assembled on IscU scaffold proteins.

In the bacterial ISC system for iron-sulfur cluster assembly, IscU acts as a primary scaffold protein, and the molecular co-chaperones HscA and HscB specifically interact with IscU to facilitate ATP-driven cluster transfer. In this work, cluster transfer from Azotobacter vinelandii [Fe(2)S(2)](2+) cluster-bound IscU to apo-Grx5, a general purpose monothiol glutaredoxin in A. vinelandii, was monitored by circular dichroism spectroscopy, in the absence and in the presence of HscA/HscB/Mg-ATP. The results indicate a 700-fold enhancement in the rate of [Fe(2)S(2)](2+) cluster transfer in the presence of the co-chaperones and Mg-ATP, yielding a second-order rate constant of 20 000 M(-1) min(-1) at 23 °C. Thus, HscA and HscB are required for efficient ATP-dependent [Fe(2)S(2)](2+) cluster transfer from IscU to Grx5. The results support a role for monothiol Grx's in storing and transporting [Fe(2)S(2)](2+) clusters assembled on IscU and illustrate the limitations of interpreting in vitro cluster transfer studies involving [Fe(2)S(2)]-IscU in the absence of the dedicated HscA/HscB co-chaperone system.

Citrullination Alters the Antiviral and Immunomodulatory Activities of the Human Cathelicidin LL-37 During Rhinovirus Infection.

Human rhinoviruses (HRV) are the most common cause of viral respiratory tract infections. While normally mild and self-limiting in healthy adults, HRV infections are associated with bronchiolitis in infants, pneumonia in immunocompromised patients, and exacerbations of asthma and COPD. The human cathelicidin LL-37 is a host defense peptide (HDP) with broad immunomodulatory and antimicrobial activities that has direct antiviral effects against HRV. However, LL-37 is known to be susceptible to the enzymatic activity of peptidyl arginine deiminases (PAD), and exposure of the peptide to these enzymes results in the conversion of positively charged arginines to neutral citrullines (citrullination). Here, we demonstrate that citrullination of LL-37 reduced its direct antiviral activity against HRV. Furthermore, while the anti-rhinovirus activity of LL-37 results in dampened epithelial cell inflammatory responses, citrullination of the peptide, and a loss in antiviral activity, ameliorates this effect. This study also demonstrates that HRV infection upregulates PAD2 protein expression, and increases levels of protein citrullination, including histone H3, in human bronchial epithelial cells. Increased PADI gene expression and HDP citrullination during infection may represent a novel viral evasion mechanism, likely applicable to a wide range of pathogens, and should therefore be considered in the design of therapeutic peptide derivatives.

Comprehensive laboratory evaluation of a specific lateral flow assay for the presumptive identification of abrin in suspicious white powders and environmental samples.

Abrin is a heterodimeric toxin present in the seeds of the Abrus precatorius plant. The easily obtainable seeds can yield a highly toxic product that can be used in various types of biocrimes and terrorism-related activities, including "white-powder" letters. Although the vast majority of these threats are hoaxes, the lack of rapid and reliable detection assays for abrin, such as lateral flow assays (LFAs), can be an impediment to accurate and rapid hazard assessment. One of the complicating factors associated with LFAs is the use of antibodies of poor affinity and specificity that cross-react with near neighbors or that bind to plant lectins, which are capable of nonspecifically cross-linking the capture and detector antibodies. Because of the critical need to promote public safety and public health, we conducted a comprehensive laboratory evaluation of a commercial LFA for the rapid detection of abrin. This study was conducted using comprehensive inclusivity and exclusivity panels of abrin and near-neighbor plant materials, along with panels of lectins, related proteins, white powders, and environmental background material, to determine the sensitivity, specificity, limit of detection, dynamic range, and repeatability of the assay for the specific intended use of evaluating suspicious white powders and environmental samples for the presumptive presence of abrin.