High mobility group box 1, ATP, lipid mediators, and tissue factor are elevated in COVID-19 patients: HMGB1 as a biomarker of worst prognosis.

The severe acute respiratory syndrome coronavirus 2, the agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, has spread worldwide since it was first identified in November 2019 in Wuhan, China. Since then, progress in pathogenesis linked severity of this systemic disease to the hyperactivation of network of cytokine-driven pro-inflammatory cascades. Here, we aimed to identify molecular biomarkers of disease severity by measuring the serum levels of inflammatory mediators in a Brazilian cohort of patients with COVID-19 and healthy controls (HCs). Critically ill patients in the intensive care unit were defined as such by dependence on oxygen supplementation (93% intubated and 7% face mask), and computed tomography profiles showing ground-glass opacity pneumonia associated to and high levels of D-dimer. Our panel of mediators included HMGB1, ATP, tissue factor, PGE2 , LTB4 , and cys-LTs. Follow-up studies showed increased serum levels of every inflammatory mediator in patients with COVID-19 as compared to HCs. Originally acting as a transcription factor, HMGB1 acquires pro-inflammatory functions following secretion by activated leukocytes or necrotic tissues. Serum levels of HMGB1 were positively correlated with cys-LTs, D-dimer, aspartate aminotransferase, and alanine aminotransferase. Notably, the levels of the classical alarmin HMGB1 were higher in deceased patients, allowing their discrimination from patients that had been discharged at the early pulmonary and hyperinflammatory phase of COVID-19. In particular, we verified that HMGB1 levels above 125.4 ng/ml is the cutoff that distinguishes patients that are at higher risk of death. In conclusion, we propose the use of serum levels of HMGB1 as a biomarker of severe prognosis of COVID-19.

Pharmacological inhibition of lysine-specific demethylase 1 (LSD1) induces global transcriptional deregulation and ultrastructural alterations that impair viability in Schistosoma mansoni.

Treatment and control of schistosomiasis still rely on only one effective drug, praziquantel (PZQ) and, due to mass treatment, the increasing risk of selecting for schistosome strains that are resistant to PZQ has alerted investigators to the urgent need to develop novel therapeutic strategies. The histone-modifying enzymes (HMEs) represent promising targets for the development of epigenetic drugs against Schistosoma mansoni. In the present study, we targeted the S. mansoni lysine-specific demethylase 1 (SmLSD1), a transcriptional corepressor, using a novel and selective synthetic inhibitor, MC3935, which was used to treat schistosomula and adult worms in vitro. By using cell viability assays and optical and electron microscopy, we showed that treatment with MC3935 affected parasite motility, egg-laying, tegument, and cellular organelle structures, culminating in the death of schistosomula and adult worms. In silico molecular modeling and docking analysis suggested that MC3935 binds to the catalytic pocket of SmLSD1. Western blot analysis revealed that MC3935 inhibited SmLSD1 demethylation activity of H3K4me1/2. Knockdown of SmLSD1 by RNAi recapitulated MC3935 phenotypes in adult worms. RNA-Seq analysis of MC3935-treated parasites revealed significant differences in gene expression related to critical biological processes. Collectively, our findings show that SmLSD1 is a promising drug target for the treatment of schistosomiasis and strongly support the further development and in vivo testing of selective schistosome LSD1 inhibitors.

The dengue vector Aedes aegypti contains a functional high mobility group box 1 (HMGB1) protein with a unique regulatory C-terminus.

The mosquito Aedes aegypti can spread the dengue, chikungunya and yellow fever viruses. Thus, the search for key molecules involved in the mosquito survival represents today a promising vector control strategy. High Mobility Group Box (HMGB) proteins are essential nuclear factors that maintain the high-order structure of chromatin, keeping eukaryotic cells viable. Outside the nucleus, secreted HMGB proteins could alert the innate immune system to foreign antigens and trigger the initiation of host defenses. In this work, we cloned and functionally characterized the HMGB1 protein from Aedes aegypti (AaHMGB1). The AaHMGB1 protein typically consists of two HMG-box DNA binding domains and an acidic C-terminus. Interestingly, AaHMGB1 contains a unique alanine/glutamine-rich (AQ-rich) C-terminal region that seems to be exclusive of dipteran HMGB proteins. AaHMGB1 is localized to the cell nucleus, mainly associated with heterochromatin. Circular dichroism analyses of AaHMGB1 or the C-terminal truncated proteins revealed α-helical structures. We showed that AaHMGB1 can effectively bind and change the topology of DNA, and that the AQ-rich and the C-terminal acidic regions can modulate its ability to promote DNA supercoiling, as well as its preference to bind supercoiled DNA. AaHMGB1 is phosphorylated by PKA and PKC, but not by CK2. Importantly, phosphorylation of AaHMGB1 by PKA or PKC completely abolishes its DNA bending activity. Thus, our study shows that a functional HMGB1 protein occurs in Aedes aegypt and we provide the first description of a HMGB1 protein containing an AQ-rich regulatory C-terminus.

Evaluation of 3-(3-chloro-phenyl)-5-(4-pyridyl)-4,5-dihydroisoxazole as a novel anti-inflammatory drug candidate.

BACKGROUND: 3-(3-chloro-phenyl)-5-(4-pyridyl)-4,5-dihydroisoxazole (DIC) is a five-membered heterocyclic compound containing a N-O bond. The anti-inflammatory effects of this compound were studied both in vitro and in vivo. PRINCIPAL FINDINGS: DIC effectively decreased TNF-α and IL-6 release from LPS-stimulated macrophages in a dose dependent manner. DIC diminished the levels of COX-2 with subsequent inhibition of PGE(2) production. DIC also compromised HMGB1 translocation from the nucleus to the cytoplasm. Moreover, DIC prevented the nuclear translocation of NF-κB and inhibited the MAPK pathway. In vivo, DIC inhibited migration of neutrophils to the peritoneal cavity of mice. CONCLUSIONS: This study presents the potential utilization of a synthetic compound, as a lead for the development of novel anti-inflammatory drugs.

CK2 phosphorylation of Schistosoma mansoni HMGB1 protein regulates its cellular traffic and secretion but not its DNA transactions.

BACKGROUND: The helminth Schistosoma mansoni parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1), a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1) is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space. PRINCIPAL FINDINGS: We showed in vitro and in vivo that CK2 phosphorylation was involved in the nucleocytoplasmic shuttling of SmHMGB1. By site-directed mutagenesis we mapped the two serine residues of SmHMGB1 that were phosphorylated by CK2. By DNA bending and supercoiling assays we showed that CK2 phosphorylation of SmHMGB1 had no effect in the DNA binding activities of the protein. We showed by electron microscopy, as well as by cell transfection and fluorescence microscopy that SmHMGB1 was present in the nucleus and cytoplasm of adult schistosomes and mammalian cells. In addition, we showed that treatments of the cells with either a phosphatase or a CK2 inhibitor were able to enhance or block, respectively, the cellular traffic of SmHMGB1. Importantly, we showed by confocal microscopy and biochemically that SmHMGB1 is significantly secreted by S. mansoni eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated. CONCLUSIONS: We showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis.

Schistosoma mansoni: SmLIMPETin, a member of a novel family of invertebrate-only regulatory proteins.

Eukaryotic LIM domain proteins contain zinc finger forming motifs rich in cysteine and histidine that enable them to interact with other proteins. A cDNA clone isolated from an adult schistosome cDNA library revealed a sequence that coded for a novel class of proteins bearing 6 LIM domains and an N-terminal PET domain, SmLIMPETin. Phylogeny reconstruction of SmLIMPETin and comparison of its sequence to invertebrate homologues and to the vertebrate four-and-a-half LIM domains protein family (FHLs), uncovered a novel LIM domain protein family, the invertebrate LIM and PET domain protein family (LIMPETin). Northern blots, RT-PCR and Western blot showed that SmLIMPETin gene was less expressed in sexually mature adult females compared to sexually immature adult females and sexually mature and immature adult males, and not expressed in schistosomula.

Cloning of a protein arginine methyltransferase PRMT1 homologue from Schistosoma mansoni: evidence for roles in nuclear receptor signaling and RNA metabolism.

The most studied arginine methyltransferase is the type I enzyme, which catalyzes the transfer of an S-adenosyl-L-methionine to a broad spectrum of substrates, including histones, RNA-transporting proteins, and nuclear hormone receptor coactivators. We cloned a cDNA encoding a protein arginine methyltransferase in Schistosoma mansoni (SmPRMT1). SmPRMT1 is highly homologous to the vertebrate PRMT1 enzyme. In vitro methylation assays showed that SmPRMT1 recombinant protein was able to specifically methylate histone H4. Two schistosome proteins likely to be involved in RNA metabolism, SMYB1 and SmSmD3, that display a number of RGG motifs, were strongly methylated by SmPRMT1. In vitro GST pull-down assays showed that SMYB1 and SmSmD3 physically interacted with SmPRMT1. Additional GST pull-down assay suggested the occurrence of a ternary complex including SmPRMT1, SmRXR1 nuclear receptor, and the p160 (SRC-1) nuclear receptor coactivator. Together, these data suggest a mechanism by which SmPRMT1 plays a role in nuclear receptor-mediated chromatin remodeling and RNA transactions.