Structural Basis of a Key Factor Regulating the Affinity between the Zonula Occludens First PDZ Domain and Claudins

The molecular seal between epithelial cells, called the tight junction (TJ), is built by several membrane proteins, with claudins playing the most prominent role. The scaffold proteins of the zonula occludens family are required for the correct localization of claudins and hence formation of the TJ. The intracellular C terminus of claudins binds to the N-terminal PDZ domain of zonula occludens proteins (PDZ1). Of the 23 identified human claudin proteins, nine possess a tyrosine at the −6 position. Here we show that the claudin affinity for PDZ1 is dependent on the presence or absence of this tyrosine and that the affinity is reduced if the tyrosine is modified by phosphorylation. The PDZ1 β2-β3 loop undergoes a significant conformational change to accommodate this tyrosine. Cell culture experiments support a regulatory role for this tyrosine. Plasticity has been recognized as a critical property of TJs that allow cell remodeling and migration. Our work provides a molecular framework for how TJ plasticity may be regulated

A mRNA Vaccine Encoding for a RBD 60-mer Nanoparticle Elicits Neutralizing Antibodies and Protective Immunity Against the SARS-CoV-2 Delta Variant in Transgenic K18-hACE2 Mice.

Two years into the COVID-19 pandemic there is still a need for vaccines to effectively control the spread of novel SARS-CoV-2 variants and associated cases of severe disease. Here we report a messenger RNA vaccine directly encoding for a nanoparticle displaying 60 receptor binding domains (RBDs) of SARS-CoV-2 that acts as a highly effective antigen. A construct encoding the RBD of the Delta variant elicits robust neutralizing antibody response, and also provides protective immunity against the Delta variant in a widely used transgenic mouse model. We ultimately find that the proposed mRNA RBD nanoparticle-based vaccine provides a flexible platform for rapid development and will likely be of great value in combatting current and future SARS-CoV-2 variants of concern

Biophysical Studies of the Interactions Between Small Molecule Inhibitors and Viral Protein Targets

Being a cause of over 4 million cases of severe illness and 300,000 deaths per year, Influenza virus is arguably one of the world largest health threats. This virus enters cells in an endosome dependent manner through the action of hemagglutinin (HA), the major regulator of viral entry process. During the endosome evolution HA undergoes a series of pH induced conformational changes that lead to the fusion of viral and endosomal membranes and the release of viral material into the cell. The central role of HA for influenza infectivity makes it a very attractive drug target. Large number of antibodies, peptides and small molecule inhibitors that block HA function has been developed over the years. However, none of the current treatment options against influenza virus infection target hemagglutinin. In this research we have chosen several small molecule inhibitors of HA-induced fusion and characterized their modes of action using a combination of structural biology and virology methods. A combination of nuclear magnetic resonance (NMR), X-ray crystallography, mutagenesis and several other approaches was applied to gain insight into the binding sites of these compounds and their inhibition mechanisms. After the detailed characterization, all obtained information was then used to navigate the chemical optimization of the select group of inhibitors

Inhibition of influenza H7 hemagglutinin-mediated entry.

The recent outbreak of H7N9 influenza in China is of high concern to public health. H7 hemagglutinin (HA) plays a critical role in influenza entry and thus HA presents an attractive target for antivirals. Previous studies have suggested that the small molecule tert-butyl hydroquinone (TBHQ) inhibits the entry of influenza H3 HA by binding to the stem loop of HA and stabilizing the neutral pH conformation of HA, thereby disrupting the membrane fusion step. Based on amino acid sequence, structure and immunogenicity, H7 is a related Group 2 HA. In this work we show, using a pseudovirus entry assay, that TBHQ inhibits H7 HA-mediated entry, as well as H3 HA-mediated entry, with an IC50 ~ 6 µM. Using NMR, we show that TBHQ binds to the H7 stem loop region. STD NMR experiments indicate that the aromatic ring of TBHQ makes extensive contact with the H7 HA surface. Limited proteolysis experiments indicate that TBHQ inhibits influenza entry by stabilizing the H7 HA neutral pH conformation. Together, this work suggests that the stem loop region of H7 HA is an attractive target for therapeutic intervention and that TBHQ, which is a widely used food preservative, is a promising lead compound

TBHQ inhibits H3 and H7 HA-mediated entry.

<p>Each concentration point was performed in triplicate.</p

Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in Cyprinidae fish: Towards hints of their arrangements using advanced classification methods

To tackle the ever-present global concern regarding human exposure to persistent organic pollutants (POPs) via food products, this study strived to indicate associations between organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in lake-fish tissue depending on the species and sampling season. Apart from the monitoring initiatives recommended in the Global Monitoring Plan for POPs, the study discussed 7 OCPs and 18 PCB congeners determined in three Cyprinidae species (rudd, carp, and Prussian carp) from Vransko Lake (Croatia), which are widely domesticated and reared as food fish across Europe and Asia. We exploit advanced classification algorithms, the Kohonen self-organizing maps (SOM) and Decision Trees (DT), to search for POP patterns typical for the investigated species. As indicated by SOM, some of the dioxin-like and non-dioxin-like PCBs (PCB-28, PCB-74, PCB-52, PCB-101, PCB-105, PCB-114, PCB-118, PCB-156 and PCB-157), alpha-HCH and beta-HCH caused dissimilarities among fish species, but regardless of their weight and length. To support these suggestions, DT analysis sequenced the fish species and seasons based on the concentration of heavier congeners. The presented assumptions indicated that the supplemental application of SOM and DT offers advantageous features over the usually rough interpretation of POPs pattern and over the single use of the methods

TBHQ stabilizes the neutral pH conformation of H7 HA.

<p>Limited proteolysis experiment for H7 HA in the presence (open squares, dotted line) and absence (filled circles, solid line) of TBHQ at different pH. The curves correspond to fits using [H⁺]_mp  = 1.30×10⁻⁵ and n = 1.6 in the absence of TBHQ and [H+]_mp  = 2.83×10⁻⁵ and n = 1.8 in the presence of TBHQ (c.f. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076363#s2" target="_blank">Experimental Procedures</a>).</p

TBHQ binds to the H3 and H7 HA stem loops.

<p>(a) WaterLOGSY NMR of TBHQ binding to H3 HA in the presence and absence of monoclonal antibody F49. (b) WaterLOGSY NMR of TBHQ binding to H7 HA in the presence and absence of monoclonal antibody F49. In (a) and (b) the aromatic resonances of TBHQ are denoted by arrows. (c) Relative STD signals of TBHQ in the presence of H3 HA. (d) Relative STD signals of TBHQ in the presence of H7 HA. The blue line represents the protein surface. The size of the red spheres represent the magnitude of the observed STD for each ¹H.</p

Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in Cyprinidae fish: Towards hints of their arrangements using advanced classification methods

To tackle the ever-present global concern regarding human exposure to persistent organic pollutants (POPs) via food products, this study strived to indicate associations between organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in lake-fish tissue depending on the species and sampling season. Apart from the monitoring initiatives recommended in the Global Monitoring Plan for POPs, the study discussed 7 OCPs and 18 PCB congeners determined in three Cyprinidae species (rudd, carp, and Prussian carp) from Vransko Lake (Croatia), which are widely domesticated and reared as food fish across Europe and Asia. We exploit advanced classification algorithms, the Kohonen self-organizing maps (SOM) and Decision Trees (DT), to search for POP patterns typical for the investigated species. As indicated by SOM, some of the dioxin-like and non-dioxin-like PCBs (PCB-28, PCB-74, PCB-52, PCB-101, PCB-105, PCB-114, PCB-118, PCB-156 and PCB-157), alpha-HCH and beta-HCH caused dissimilarities among fish species, but regardless of their weight and length. To support these suggestions, DT analysis sequenced the fish species and seasons based on the concentration of heavier congeners. The presented assumptions indicated that the supplemental application of SOM and DT offers advantageous features over the usually rough interpretation of POPs pattern and over the single use of the methods

Discovery of Selective Inhibitors of the <i>Clostridium difficile</i> Dehydroquinate Dehydratase

<div><p>A vibrant and healthy gut flora is essential for preventing the proliferation of <i>Clostridium difficile</i>, a pathogenic bacterium that causes severe gastrointestinal symptoms. In fact, most <i>C. difficile</i> infections (CDIs) occur after broad-spectrum antibiotic treatment, which, by eradicating the commensal gut bacteria, allows its spores to proliferate. Hence, a <i>C. difficile</i> specific antibiotic that spares the gut flora would be highly beneficial in treating CDI. Towards this goal, we set out to discover small molecule inhibitors of the <i>C. difficile</i> enzyme dehydroquinate dehydratase (DHQD). DHQD is the 3^rd of seven enzymes that compose the shikimate pathway, a metabolic pathway absent in humans, and is present in bacteria as two phylogenetically and mechanistically distinct types. Using a high-throughput screen we identified three compounds that inhibited the type I <i>C. difficile</i> DHQD but not the type II DHQD from <i>Bacteroides thetaiotaomicron</i>, a highly represented commensal gut bacterial species. Kinetic analysis revealed that the compounds inhibit the <i>C. difficile</i> enzyme with K_i values ranging from 10 to 20 µM. Unexpectedly, kinetic and biophysical studies demonstrate that inhibitors also exhibit selectivity between type I DHQDs, inhibiting the <i>C. difficile</i> but not the highly homologous <i>Salmonella enterica</i> DHQD. Therefore, the three identified compounds seem to be promising lead compounds for the development of <i>C. difficile</i> specific antibiotics.</p></div