Structural and Functional Consequences Induced by Post-Translational Modifications in α-Defensins

HNP-1 is an antimicrobial peptide that undergoes proteolytic cleavage to become a mature peptide. This process represents the mechanism commonly used by the cells to obtain a fully active antimicrobial peptide. In addition, it has been recently described that HNP-1 is recognized as substrate by the arginine-specific ADP-ribosyltransferase-1. Arginine-specific mono-ADP-ribosylation is an enzyme-catalyzed post-translational modification in which NAD+ serves as donor of the ADP-ribose moiety, which is transferred to the guanidino group of arginines in target proteins. While the arginine carries one positive charge, the ADP-ribose is negatively charged at the phosphate moieties at physiological pH. Therefore, the attachment of one or more ADP-ribose units results in a marked change of cationicity. ADP-ribosylation of HNP-1 drastically reduces its cytotoxic and antibacterial activities. While the chemotactic activity of HNP-1 remains unaltered, its ability to induce interleukin-8 production is enhanced. The arginine 14 of HNP-1 modified by the ADP-ribose is in some cases processed into ornithine, perhaps representing a different modality in the regulation of HNP-1 activities

NAD-dependent ADP-ribosylation of the human antimicrobial and immune-modulatory peptide LL-37 by ADP-ribosyltransferase-1

LL-37 is a cationic peptide belonging to the cathelicidin family that has antimicrobial and immune-modulatory properties. Here we show that the mammalian mono-ADP-ribosyltransferase-1 (ART1), which selectively transfers the ADP-ribose moiety from NAD to arginine residues, ADP-ribosylates LL-37 in vitro. The incorporation of ADP-ribose was first observed by Western blot analysis and then confirmed by MALDI-TOF. Mass-spectrometry showed that up to four of the five arginine residues present in LL-37 could be ADP-ribosylated on the same peptide when incubated at a high NAD concentration in the presence of ART1. The attachment of negatively charged ADP-ribose moieties considerably alters the positive charge of the arginine residues thus reducing the cationicity of LL-37. The cationic nature of LL-37 is key for its ability to interact with cell membranes or negatively charged biomolecules, such as DNA, RNA, F-actin and glycosaminoglycans. Thus, the ADP-ribosylation of LL-37 is expected to have the potential to modulate LL-37 biological activities in several physiological and pathological settings

Auto ADP-ribosylation of NarE, a Neisseria meningitidis ADP-ribosyltransferase, regulates its catalytic activities

NarE is an arginine-specific mono-ADPribosyltransferase identified in Neisseria meningitidis that requires the presence of iron in a structured cluster for its enzymatic activities. In this study, we show that NarE can perform auto-ADP-ribosylation. This automodification occurred in a time- and NADconcentration- dependent manner; was inhibited by novobiocin, an ADP-ribosyltransferase inhibitor; and did not occur when NarE was heat inactivated. No reduction in incorporation was evidenced in the presence of high concentrations of ATP, GTP, ADP-ribose, or nicotinamide, which inhibits NAD-glycohydrolase, impeding the formation of free ADP-ribose. Based on the electrophoretic profile of NarE on auto-ADP-ribosylation and on the results of mutagenesis and mass spectrometry analysis, the auto-ADP-ribosylation appeared to be restricted to the addition of a single ADP-ribose. Chemical stability experiments showed that the ADP-ribosyl linkage was sensitive to hydroxylamine, which breaks ADP-ribose-arginine bonds. Sitedirected mutagenesis suggested that the auto-ADP-ribosylation site occurred preferentially on the R7 residue, which is located in the region I of the ADP-ribosyltransferase family. After auto-ADP-ribosylation, NarE showed a reduction in ADP-ribosyltransferase activity, while NAD-glycohydrolase activity was increased. Overall, our findings provide evidence for a novel intramolecular mechanism used by NarE to regulate its enzymatic activities.—Picchianti, M., Del Vecchio, M., Di Marcello, F., Biagini, M., Veggi, D., Norais N., Rappuoli, R., Pizza, M., Balducci, E. Auto ADP-ribosylation of NarE, a Neisseria meningitidis ADP-ribosyltransferase, regulates its catalytic activities. FASEB J

STUDIO OSSERVAZIONALE SULL'UTILIZZO OFF-LABEL DEL BARICITINIB PER IL TRATTAMENTO DI SOGGETTI ADULTI OSPEDALIZZATI CON COVID-19 GRAVE

A dicembre 2019 in Cina, è stata individuata un infezione causata dal virus Sars-Cov-2, appartenente alla famiglia dei coronavirus. La sindrome clinica provocata da Sars CoV-2 è caratterizzata dai seguenti sintomi : febbre, tosse e affaticamento, produzione di espettorato, mal di testa, emottisi, diarrea, dispnea, e linfopenia. In una minoranza di casi (circa 5-6% dei casi) invece la malattia può manifestarsi in forma moderata o grave con rischio di complicanze soprattutto respiratorie (insufficienza respiratoria, ARDS). Nei casi più gravi può verificarsi una polmonite, una sindrome da distress respiratorio acuto, sepsi e uno shock settico fino ad arrivare al decesso del paziente. I pazienti con sintomi più gravi, mostrano livelli più elevati di espressione del recettore dell'interleuchina-2 (IL-2) e dell'interleuchina-6 (IL-6). Il farmaco Baricitinib (Olumiant®) è un inibitore selettivo e reversibile di Janus Chinasi (JAK1 e JAK2), che fosforilano ed attivano trasduttori di segnale e attivatori della trascrizione (STAT) con conseguente espressione di citochine e di fattori di crescita coinvolti nella ematopoiesi. Lo studio REGBAR-00 ha dimostrato l’efficacia e la sicurezza del trattamento farmacologico con baricitinib in pazienti affetti da polmonite da COVID-19, con iniziale insufficienza respiratoria che nella attuale pratica clinica evolvono, in percentuali elevate, ad un supporto della funzione respiratoria meccanica invasiva o non invasiva o decesso

Arginine-specific mono ADP-ribosylation in vitro of antimicrobial peptides by ADP-ribosylating toxins.

Among the several toxins used by pathogenic bacteria to target eukaryotic host cells, proteins that exert ADP-ribosylation activity represent a large and studied family of dangerous and potentially lethal toxins. These proteins alter cell physiology catalyzing the transfer of the ADP-ribose unit from NAD to cellular proteins involved in key metabolic pathways. In the present study, we tested the capability of four of these toxins, to ADP-ribosylate α- and β- defensins. Cholera toxin (CT) from Vibrio cholerae and heat labile enterotoxin (LT) from Escherichia coli both modified the human α-defensin (HNP-1) and β- defensin-1 (HBD1), as efficiently as the mammalian mono-ADP-ribosyltransferase-1. Pseudomonas aeruginosa exoenzyme S was inactive on both HNP-1 and HBD1. Neisseria meningitidis NarE poorly recognized HNP-1 as a substrate but it was completely inactive on HBD1. On the other hand, HNP-1 strongly influenced NarE inhibiting its transferase activity while enhancing auto-ADP-ribosylation. We conclude that only some arginine-specific ADP-ribosylating toxins recognize defensins as substrates in vitro. Modifications that alter the biological activities of antimicrobial peptides may be relevant for the innate immune response. In particular, ADP-ribosylation of antimicrobial peptides may represent a novel escape mechanism adopted by pathogens to facilitate colonization of host tissues

Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197

CRM197 is an enzymatically inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution (G52E). Being naturally nontoxic, CRM197 is an ideal carrier protein for conjugate vaccines against encapsulated bacteria and is currently used to vaccinate children globally against Haemophilus influenzae, pneumococcus, and meningococcus. To understand the molecular basis for lack of toxicity in CRM197, we determined the crystal structures of the full-length nucleotide-free CRM197 and of CRM197 in complex with the NAD hydrolysis product nicotinamide (NCA), both at 2.0-Å resolution. The structures show for the first time that the overall fold of CRM197 and DT are nearly identical and that the striking functional difference between the two proteins can be explained by a flexible active-site loop that covers the NAD binding pocket. We present the molecular basis for the increased flexibility of the active-site loop in CRM197 as unveiled by molecular dynamics simulations. These structural insights, combined with surface plasmon resonance, NAD hydrolysis, and differential scanning fluorimetry data, contribute to a comprehensive characterization of the vaccine carrier protein, CRM197

HNP-1 is ADP-ribosylated at R14.

<p>HNP-1 R14K and HNP-1 R15K protein variants (3 µg, 43.85 µM) were incubated with CTA (2.5 U), LTA (8.9 U) or ART1 (5.5 U) in the presence of 10 µM biotin-NAD, in 50 mM potassium phosphate buffer, pH 7.5 at 30°C for 1 h. The ADP-ribosylated peptides were separated by SDS-PAGE in a 10% NuPAGE gel and transferred to nitrocellulose. Membranes treated as previously described, were incubated with streptavidin-HRP conjugated (1∶10000 dilution) before visualization of the biotin-ADP-ribose labeled bands by chemiluminescence. Here shown in comparison with the modification of HNP-1 wild-type in the same reaction conditions.</p

Modification of

<p>β<b>-defensin by selected ADP-ribosyltransferases.</b> (<b>A</b>) HBD1 is ADP-ribosylated by CTA and LTA. HBD1 (3 µg, 38.18 µM)) was incubated with CTA (2.5 U) or LTA (8.9 U) in the presence of 10 µM biotin-NAD, in 50 mM potassium phosphate buffer, pH 7.5 at 30°C for 1 h (Toxin). Reactions were also performed in the presence of 2 mM NAD (Toxin + NAD) or 2 mM ADP-ribose (Toxin + ADP-ribose). Control reactions performed with heat-inactivated CTA or LTA (HI-Toxin) or in the absence of toxins (-Toxin) are also shown. The ADP-ribosylated peptides were separated by SDS-PAGE in a 10% NuPAGE gel and transferred to nitrocellulose. The membrane was treated as previously described, incubated with streptavidin-HRP conjugated (1∶10000 dilution) before visualization of the biotin-ADP-ribose labeled bands by chemiluminescence. (<b>B</b>) ADP-ribosylation of HBD1 by ART1. HBD1 (3 µg, 38.18 µM) was incubated with 6.8 U of ART1 (ART1) or heat-inactivated ART1 (HI-ART1) and 10 µM biotin-NAD in 50 mM potassium phosphate buffer, pH 7.5, at 30°C for 1 h. (<b>C</b>) HBD1 is ADP-ribosylated in a dose response fashion. HBD1 at the concentration shown in the Figure was incubated with CTA (2.5 U) or LTA (8.9 U) in the presence of 10 µM biotin-NAD, in 50 mM potassium phosphate buffer, pH 7.5 at 30°C for 1 h. (<b>D</b>) Time dependent ADP-ribosylation of HBD1. HBD1 (3 µg) was incubated with CTA (1.25 U) or LTA (4.45 U) using the same conditions above described. Times of incubation are indicated in the Figure. Molecular markers are on the left. Data shown are representative of two independent experiments.</p