ALX/FPR2 Activation by Stereoisomers of D1 Resolvins Elucidating with Molecular Dynamics Simulation.

Chronic inflammation contributes to several diseases, but its resolution is driven by specialized pro-resolving mediators (SPM) such as resolvin D1 (RvD1) and its epimer aspirin-triggered resolvin D1 (AT-RvD1), both biosynthesized from ω-3 fatty docosahexaenoic acid (DHA). RvD1 and AT-RvD1 have anti-inflammatory and pro-resolution potentials, and their effects could be mediated by formyl peptide receptor type 2 receptor ALX/FPR2, a G-protein-coupled receptor (GPCR). In this work, we performed 44 µs of molecular dynamics simulations with two complexes: FPR2@AT-RvD1 and FPR2@RvD1. Our results show the following: (i) in the AT-RvD1 simulations, the ALX/FPR2 receptor remained in the active state in 62% of the frames, while in the RVD1 simulations, the receptor remained in the active state in 74% of the frames; (ii) two residues, R201 and R205, of ALX/FPR2 appear, establishing interactions with both resolvins in all simulations (22 in total); (iii) RvD1 hydrogen bonds with R201 and R205 presented higher frequency than AT-RvD1; and (iv) residues R201 and R205 are the two receptor hotspots, demonstrated by the binding free calculations. Such results show that the ALX/FPR2 receptor remained in the active state for longer in the FPR2@RvD1 simulations than in the FPR2@AT-RvD1 simulations.

Antivirals virtual screening to SARS-CoV-2 non-structural proteins.

In March 2020, the World Health Organization (WHO) declared coronavirus disease-19 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a pandemic. Since then, the search for a vaccine or drug for COVID-19 treatment has started worldwide. In this regard, a fast approach is the repurposing of drugs, primarily antiviral drugs. Herein, we performed a virtual screening using 22 antiviral drugs retrieved from the DrugBank repository, azithromycin (antibiotic), ivermectin (antinematode), and seven non-structural proteins (Nsps) of SARS-CoV-2, which are considered important targets for drugs, via molecular docking and molecular dynamics simulations. Drug-receptor binding energy was employed as the main descriptor. Based on the results, paritaprevir was predicted as a promising multi-target drug that favorably bound to all tested Nsps, mainly adipose differentiation-related protein (ADRP) (-36.2 kcal mol-1) and coronavirus main proteinase (Mpro) (-32.2 kcal mol-1). Moreover, the results suggest that simeprevir is a strong inhibitor of Mpro (-37.2 kcal mol-1), which is an interesting finding because Mpro plays an important role in viral replication. In addition to drug-receptor affinity, hot spot residues were characterized to facilitate the design of new drug derivatives with improved biological responses.

DISTINCT PHENOTYPE OF NON-ALCOHOLIC FATTY LIVER DISEASE IN PATIENTS WITH LOW LEVELS OF FREE COPPER AND OF CERULOPLASMIN.

BACKGROUND: Copper deficiency has been linked to alterations in lipid metabolism and hepatic steatosis. Oxidative stress plays a role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). One of the enzymes that neutralize oxidative stress is Cu/Zn superoxide dismutase, which depends on the availability of adequate amounts of copper. OBJECTIVE: Correlate the levels of ceruloplasmin and of non-ceruloplasmin-bound copper (NCBC) with clinical, biochemical and histological parameters of non-alcoholic fatty liver disease (NAFLD) patients. METHODS: Data from 95 consecutively admitted NAFLD patients who underwent liver biopsy composed the groups based on ceruloplasmin levels lower than 25 mg/dL and on negative NCBC. The risk factors for NAFLD in each group were compared. RESULTS: Body mass index was lower in patients with ceruloplasmin <25 mg/dL (29.1±3.47 vs 32.8±6.24 kg/m2; P=0.005) as were the levels of LDL, HDL and total cholesterol, when compared with their counterparts with ceruloplasmin >25 mg/dL (101±38 vs 116±35 mg/dL, P=0.05; 43±9 vs 51±16 mg/dL, P=0.01; 174±43 vs 197±39 mg/dL, P=0.01, respectively). Mean serum ferritin levels were higher in the ceruloplasmin <25 mg/dL group (343±327 vs 197±190 ng/mL; P=0.02). Otherwise, patients with negative NCBC had higher HOMA-IR (8.2±14.7 vs 4.6±3.7; P=0.03). Age, gender, hypertension and diabetes showed no statistical difference. CONCLUSION: Patients with NAFLD had different clinical and biochemical markers according to the levels of NCBC and ceruloplasmin.

Fenótipo distinto da doença hepática gordurosa não alcoólica em pacientes com níveis reduzidos de cobre livre e ceruloplasmina / Distinct phenotype of non-alcoholic fatty liver disease in patients with low levels of free copper and of ceruloplasmin

ABSTRACT BACKGROUND: Copper deficiency has been linked to alterations in lipid metabolism and hepatic steatosis. Oxidative stress plays a role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). One of the enzymes that neutralize oxidative stress is Cu/Zn superoxide dismutase, which depends on the availability of adequate amounts of copper. OBJECTIVE: Correlate the levels of ceruloplasmin and of non-ceruloplasmin-bound copper (NCBC) with clinical, biochemical and histological parameters of non-alcoholic fatty liver disease (NAFLD) patients. METHODS: Data from 95 consecutively admitted NAFLD patients who underwent liver biopsy composed the groups based on ceruloplasmin levels lower than 25 mg/dL and on negative NCBC. The risk factors for NAFLD in each group were compared. RESULTS: Body mass index was lower in patients with ceruloplasmin <25 mg/dL (29.1±3.47 vs 32.8±6.24 kg/m2; P=0.005) as were the levels of LDL, HDL and total cholesterol, when compared with their counterparts with ceruloplasmin >25 mg/dL (101±38 vs 116±35 mg/dL, P=0.05; 43±9 vs 51±16 mg/dL, P=0.01; 174±43 vs 197±39 mg/dL, P=0.01, respectively). Mean serum ferritin levels were higher in the ceruloplasmin <25 mg/dL group (343±327 vs 197±190 ng/mL; P=0.02). Otherwise, patients with negative NCBC had higher HOMA-IR (8.2±14.7 vs 4.6±3.7; P=0.03). Age, gender, hypertension and diabetes showed no statistical difference. CONCLUSION: Patients with NAFLD had different clinical and biochemical markers according to the levels of NCBC and ceruloplasmin.

RESUMO CONTEXTO: A deficiência de cobre tem sido relacionada a alterações no metabolismo lipídico e esteatose hepática. O estresse oxidativo desempenha um papel fundamental na fisiopatologia da doença hepática gordurosa não alcoólica. Uma das enzimas que neutralizam o estresse oxidativo é a Cobre/Zinco superoxido dismutase, que depende da disponibilidade de quantidades adequadas de cobre. OBJETIVO: Correlacionar os níveis de ceruloplasmina e de cobre não ligado à ceruloplasmina (NCBC) com parâmetros clínicos, bioquímicos e histológicos de pacientes com doença hepática gordurosa não alcoólica (DHGNA). MÉTODOS: Dados de 95 pacientes com DHGNA internados consecutivamente e submetidos à biópsia hepática compuseram os grupos com base em níveis de ceruloplasmina inferiores a 25 mg/dL e em NCBC negativo. Os fatores de risco para DHGNA em cada grupo foram comparados. RESULTADOS: O índice de massa corporal foi menor nos pacientes com ceruloplasmina <25 mg/dL (29,1±3,47 vs 32,8±6,24 kg/m2; P=0,005), assim como os níveis de LDL, HDL e colesterol total, quando comparados aos seus pares com ceruloplasmina >25 mg/dL (101±38 vs 116±35 mg/dL, P=0,05; 43±9 vs 51±16 mg/dL, P=0,01; 174±43 vs 197±39 mg/dL, P=0,01, respectivamente). Os níveis médios de ferritina sérica foram maiores no grupo ceruloplasmina <25 mg/dL (343±327 vs 197±190 mg/mL; P=0,02). Os pacientes com NCBC negativo apresentaram maior HOMA-IR (8,2±14,7 vs 4,6±3,7; P=0,03). Idade, sexo, hipertensão e diabetes não mostraram diferença estatística. CONCLUSÃO: Pacientes com DHGNA apresentaram diferentes marcadores clínicos e bioquímicos de acordo com os níveis de NCBC e ceruloplasmina.

The putative Leishmania telomerase RNA (LeishTER) undergoes trans-splicing and contains a conserved template sequence.

Telomerase RNAs (TERs) are highly divergent between species, varying in size and sequence composition. Here, we identify a candidate for the telomerase RNA component of Leishmania genus, which includes species that cause leishmaniasis, a neglected tropical disease. Merging a thorough computational screening combined with RNA-seq evidence, we mapped a non-coding RNA gene localized in a syntenic locus on chromosome 25 of five Leishmania species that shares partial synteny with both Trypanosoma brucei TER locus and a putative TER candidate-containing locus of Crithidia fasciculata. Using target-driven molecular biology approaches, we detected a ∼2,100 nt transcript (LeishTER) that contains a 5' spliced leader (SL) cap, a putative 3' polyA tail and a predicted C/D box snoRNA domain. LeishTER is expressed at similar levels in the logarithmic and stationary growth phases of promastigote forms. A 5'SL capped LeishTER co-immunoprecipitated and co-localized with the telomerase protein component (TERT) in a cell cycle-dependent manner. Prediction of its secondary structure strongly suggests the existence of a bona fide single-stranded template sequence and a conserved C[U/C]GUCA motif-containing helix II, representing the template boundary element. This study paves the way for further investigations on the biogenesis of parasite TERT ribonucleoproteins (RNPs) and its role in parasite telomere biology.

LmHus1 is required for the DNA damage response in Leishmania major and forms a complex with an unusual Rad9 homologue.

Genotoxic stress activates checkpoint-signalling pathways leading to cell cycle arrest and DNA repair. In many eukaryotes, the Rad9-Hus1-Rad1 (9-1-1) checkpoint complex participates in the early steps of the DNA damage response to replicative stress and is a pivotal contributor to genome homeostasis. The remarkable genome plasticity of the protozoan Leishmania hints at a peculiar DNA metabolism in these ancient eukaryotes. Therefore, we set out to investigate the existence of homologues of the 9-1-1 components in Leishmania major and found that LmHus1 and LmRad9 are phylogenetically related to the 9-1-1 complex subunits from other eukaryotes. Altered levels of LmHus1 and LmRad9 affected the parasite ability to manage genotoxic stress and LmHus1-defficent cells were defective in controlling cell cycle progression in response to genotoxic stress. Upon DNA damage, LmHus1 was recruited to the chromatin and colocalized with the single-stranded DNA-binding protein LmRpa1. Also, LmHus1 interacted with LmRad9 to form a DNA damage responsive complex in vivo. Altogether, our data strongly indicate the participation of LmHus1, LmRad9 and LmRpa1 in the L. major DNA damage response and suggest their involvement in genome maintenance mechanisms.

The Hus1 homologue of Leishmania major encodes a nuclear protein that participates in DNA damage response.

The protozoan parasite Leishmania presents a dynamic and plastic genome in which gene amplification and chromosome translocations are common phenomena. Such plasticity hints at the necessity of dependable genome maintenance pathways. Eukaryotic cells have evolved checkpoint control systems that recognize altered DNA structures and halt cell cycle progression allowing DNA repair to take place. In these cells, the PCNA-related heterotrimeric complex formed by the proteins Hus1, Rad9, and Rad1 is known to participate in the early steps of replicative stress sensing and signaling. Here we show that the Hus1 homolog of Leishmania major is a nuclear protein that improves the cell capability to cope with replicative stress. Overexpression of LmHus1 confers resistance to the genotoxic drugs hydroxyurea (HU) and methyl methanesulfonate (MMS) and resistance to HU correlates to reduced net DNA damage upon LmHus1 expression.