Zinc Binding Properties of Engineered RING Finger Domain of Arkadia E3 Ubiquitin Ligase

Human Arkadia is a nuclear protein consisted of 989 amino acid residues, with a characteristic RING domain in its C-terminus. The RING domain harbours the E3 ubiquitin ligase activity needed by Arkadia to ubiquitinate its substrates such as negative regulators of TGF-β signaling. The RING finger domain of Arkadia is a RING-H2 type and its structure and stability is strongly dependent on the presence of two bound Zn(II) ions attached to the protein frame through a defined Cys3-His2-Cys3 motif. In the present paper we transform the RING-H2 type of Arkadia finger domain to nonnative RING sequence, substituting the zinc-binding residues Cys955 or His960 to Arginine, through site-directed mutagenesis. The recombinant expression, in Escherichia coli, of the mutants C955R and H960R reveal significant lower yield in respect with the native polypeptide of Arkadia RING-H2 finger domain. In particular, only the C955R mutant exhibits expression yield sufficient for recombinant protein isolation and preliminary studies. Atomic absorption measurements and preliminary NMR data analysis reveal that the C955R point mutation in the RING Finger domain of Arkadia diminishes dramatically the zinc binding affinity, leading to the breakdown of the global structural integrity of the RING construct

A NMR study of the interaction of a three-domain construct of ATP7A with copper(I) and copper(I)-HAH1: the interplay of domains.

ATP7A is a P-type ATPase involved in copper(I) homeostasis in humans. It possesses a long N-terminal tail protruding into the cytosol and containing six copper(I)-binding domains, which are individually folded and capable of binding one copper(I) ion. ATP7A receives copper from a soluble protein, the metallochaperone HAH1. The exact role and interplay of the six soluble domains is still quite unclear, as it has been extensively demonstrated that they are strongly redundant with respect to copper(I) transport in vivo. In the present work, a three-domain (fourth to sixth, MNK456) construct has been investigated in solution by NMR, in the absence and presence of copper(I). In addition, the interaction of MNK456 with copper(I)-HAH1 has been studied. It is proposed that the fourth domain is the preferential site for the initial interaction with the partner. A significant dependence of the overall domain dynamics on the metallation state and on the presence of HAH1 is observed. This dependence could constitute the molecular mechanism to trigger copper(I) translocation and/or ATP7A relocalization from the trans-Golgi network to the plasmatic membrane

Analytical and in silico study of the inclusion complexes between tropane alkaloids atropine and scopolamine with cyclodextrins

AbstractDatura stramonium L. (Solanaceae) possesses a rich tropane alkaloids (TAs) spectrum. The plant contains, in particular, the allelopathic compounds scopolamine and atropine, which are poorly soluble in water, thus limiting their use in agrochemical formulations as biocidal and deterrent agents against herbivore insects. The efficacy of the hydrophobic TAs extracts could be increased with the improvement of their dissolution/leaching properties. This is important for improving screening and test performance and for elucidating the activity of environmentally friendly agricultural approaches, with new perspectives for the production and use of those biodegradable insecticidal products. The present study explores the aspects of atropine and scopolamine complexation with cyclodextrin (CDs) through FT-IR and UV–Vis spectroscopies. In addition, the structures of the inclusion complex of atropine, scopolamine and β-CD have been investigated by molecular modeling techniques. The results obtained indicate that β-CDs are a promising carriers for improving the properties of TAs, therefore increasing their application potential in agrochemical formulations. Graphic abstrac

Organotin(IV) Derivatives of L-Cysteine and their in vitro Anti-Tumor Properties

The synthesis and characterization of the organotin compounds [(n-C4H9)2Sn(cys)] (1), [(C6H5)2Sn(cys)] (2), [(C6H5)3Sn(Hcys).(H2o)] (3), {[(CH3)2Sn(Kcys)2].2(H20)} (4), {[(n-C4H9)2Sn(Kcys)2].2(H20)} (5) and {[(C6H5)2Sn(Kcys)2].2(H20)} (6) (where H2cys = L-cysteine) are reported. The compounds have been characterized by elemental analysis and 1H-NMR, Uv-Vis, FT-IR and MOssbauer spectroscopic techniques. Attempted recrystallization of (2) in DMSO/methanol 2:1 solution yielded after several days unexpectedly the dimeric compound bis(tri-phenyltin)sulphide {[(C6H5)3Sn]2S} (7) which has been characterized by x-ray analysis. The structure of the parent complex (2) as well as the mechanism of the decomposition of cysteine are being further investigated. The in vitro anticancer activity of complexes (I)- (6), against human leukemia (HL60), human liver (Bel7402), human stomach (BGC823) and human cervix epithelial human carcinoma (Hela), nasopharyngeal carcinoma (KB) and lung cancer (PG) tumor cells, were evaluated

Putative antimicrobial peptides within bacterial proteomes affect bacterial predominance: a network analysis perspective

The predominance of bacterial taxa in the gut, was examined in view of the putative antimicrobial peptide sequences (AMPs) within their proteomes. The working assumption was that compatible bacteria would share homology and thus immunity to their putative AMPs, while competing taxa would have dissimilarities in their proteome-hidden AMPs. A network–based method (“Bacterial Wars”) was developed to handle sequence similarities of predicted AMPs among UniProt-derived protein sequences from different bacterial taxa, while a resulting parameter (“Die” score) suggested which taxa would prevail in a defined microbiome. T he working hypothesis was examined by correlating the calculated Die scores, to the abundance of bacterial taxa from gut microbiomes from different states of health and disease. Eleven publicly available 16S rRNA datasets and a dataset from a full shotgun metagenomics served for the analysis. The overall conclusion was that AMPs encrypted within bacterial proteomes affected the predominance of bacterial taxa in chemospheres

Discovery of a new generation of angiotensin receptor blocking drugs:Receptor mechanisms and in silico binding to enzymes relevant to SARS-CoV-2

The discovery and facile synthesis of a new class of sartan-like arterial antihypertensive drugs (angiotensin receptor blockers [ARBs]), subsequently referred to as “bisartans” is reported. In vivo results and complementary molecular modelling presented in this communication indicate bisartans may be beneficial for the treatment of not only heart disease, diabetes, renal dysfunction, and related illnesses, but possibly COVID-19. Bisartans are novel bis-alkylated imidazole sartan derivatives bearing dual symmetric anionic biphenyl tetrazole moieties. In silico docking and molecular dynamics studies revealed bisartans exhibited higher binding affinities for the ACE2/spike protein complex (PDB 6LZG) compared to all other known sartans. They also underwent stable docking to the Zn2+ domain of the ACE2 catalytic site as well as the critical interfacial region between ACE2 and the SARS-CoV-2 receptor binding domain. Additionally, semi-stable docking of bisartans at the arginine-rich furin-cleavage site of the SARS-CoV-2 spike protein (residues 681–686) required for virus entry into host cells, suggest bisartans may inhibit furin action thereby retarding viral entry into host cells. Bisartan tetrazole groups surpass nitrile, the pharmacophoric “warhead” of PF-07321332, in its ability to disrupt the cysteine charge relay system of 3CLpro. However, despite the apparent targeting of multifunctional sites, bisartans do not inhibit SARS-CoV-2 infection in bioassays as effectively as PF-07321332 (Paxlovid)

Molecular epidemiology of SARS-CoV-2: the dominant role of arginine in mutations and infectivity

Background, Aims, Methods, Results, Conclusions: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global challenge due to its ability to mutate into variants that spread more rapidly than the wild-type virus. The molecular biology of this virus has been extensively studied and computational methods applied are an example paradigm for novel antiviral drug therapies. The rapid evolution of SARS-CoV-2 in the human population is driven, in part, by mutations in the receptor-binding domain (RBD) of the spike (S-) protein, some of which enable tighter binding to angiotensin-converting enzyme (ACE2). More stable RBD-ACE2 association is coupled with accelerated hydrolysis by proteases, such as furin, trypsin, and the Transmembrane Serine Protease 2 (TMPRSS2) that augment infection rates, while inhibition of the 3-chymotrypsin-like protease (3CLpro) can prevent the viral replication. Additionally, non-RBD and non-interfacial mutations may assist the S-protein in adopting thermodynamically favorable conformations for stronger binding. This study aimed to report variant distribution of SARS-CoV-2 across European Union (EU)/European Economic Area (EEA) countries and relate mutations with the driving forces that trigger infections. Variants’ distribution data for SARS-CoV-2 across EU/EEA countries were mined from the European Centre for Disease Prevention and Control (ECDC) based on the sequence or genotyping data that are deposited in the Global Science Initiative for providing genomic data (GISAID) and The European Surveillance System (TESSy) databases. Docking studies performed with AutoDock VINA revealed stabilizing interactions of putative antiviral drugs, e.g., selected anionic imidazole biphenyl tetrazoles, with the ACE2 receptor in the RBD-ACE2 complex. The driving forces of key mutations for Alpha, Beta, Gamma, Delta, Epsilon, Kappa, Lambda, and Omicron variants, which stabilize the RBD-ACE2 complex, were investigated by computational approaches. Arginine is the critical amino acid in the polybasic furin cleavage sites S1/S2 (681-PRRARS-686) S2′ (814-KRS-816). Critical mutations into arginine residues that were found in the delta variant (L452R, P681R) and may be responsible for the increased transmissibility and morbidity are also present in two widely spreading omicron variants, named BA.4.6 and BQ.1, where mutation R346T in the S-protein potentially contributes to neutralization escape. Arginine binders, such as Angiotensin Receptor Blockers (ARBs), could be a class of novel drugs for treating COVID-19

Probing Conformational Dynamics by Protein Contact Networks: Comparison with NMR Relaxation Studies and Molecular Dynamics Simulations

Protein contact networks (PCNs) have been used for the study of protein structure and function for the past decade. In PCNs, each amino acid is considered as a node while the contacts among amino acids are the links/edges. We examined the possible correlation between the closeness centrality measure of amino acids within PCNs and their mobility as known from NMR spin relaxation experiments and molecular dynamic (MD) simulations. The pivotal observation was that plasticity within a protein stretch correlated inversely to closeness centrality. Effects on protein conformational plasticity caused by the formation of disulfide bonds or protein–protein interactions were also identified by the PCN analysis measure closeness centrality and the hereby introduced percentage of closeness centrality perturbation (% CCP). All the comparisons between PCN measures, NMR data, and MDs were performed in a set of proteins of different biological functions and structures: the core protease domain of anthrax lethal factor, the N-terminal RING domain of E3 Ub ligase Arkadia, the reduced and oxidized forms of human thioredoxin 1, and the ubiquitin molecules (Ub) of the catalytic Ub–RING–E3–E2–Ub complex of E3 ligase Ark2.The graph theory analysis of PCNs could thus provide a general method for assessing the conformational dynamics of free proteins and putative plasticity changes between different protein forms (apo/complexed or reduced/oxidized)

Structural Identification of Metalloproteomes in Marine Diatoms, an Efficient Algae Model in Toxic Metals Bioremediation

The biosorption of pollutants using microbial organisms has received growing interest in the last decades. Diatoms, the most dominant group of phytoplankton in oceans, are (i) pollution tolerant species, (ii) excellent biological indicators of water quality, and (iii) efficient models in assimilation and detoxification of toxic metal ions. Published research articles connecting proteomics with the capacity of diatoms for toxic metal removal are very limited. In this work, we employed a structural based systematic approach to predict and analyze the metalloproteome of six species of marine diatoms: Thalassiosira pseudonana, Phaeodactylum tricornutum, Fragilariopsis cylindrus, Thalassiosira oceanica, Fistulifera solaris, and Pseudo-nitzschia multistriata. The results indicate that the metalloproteome constitutes a significant proportion (~13%) of the total diatom proteome for all species investigated, and the proteins binding non-essential metals (Cd, Hg, Pb, Cr, As, and Ba) are significantly more than those identified for essential metals (Zn, Cu, Fe, Ca, Mg, Mn, Co, and Ni). These findings are most likely related to the well-known toxic metal tolerance of diatoms. In this study, metalloproteomes that may be involved in metabolic processes and in the mechanisms of bioaccumulation and detoxification of toxic metals of diatoms after exposure to toxic metals were identified and described