Crystal structure of human S100A8 in complex with zinc and calcium

Background

S100 proteins are a large family of calcium binding proteins present only in vertebrates. They function intra- and extracellularly both as regulators of homeostatic processes and as potent effectors during inflammation. Among these, S100A8 and S100A9 are two major constituents of neutrophils that can assemble into homodimers, heterodimers and higher oligomeric species, including fibrillary structures found in the ageing prostate. Each of these forms assumes specific functions and their formation is dependent on divalent cations, notably calcium and zinc. In particular, zinc appears as a major regulator of S100 protein function in a disease context. Despite this central role, no structural information on how zinc bind to S100A8/S100A9 and regulates their quaternary structure is yet available.

Results

Here we report two crystallographic structures of calcium and zinc-loaded human S100A8. S100A8 binds two zinc ions per homodimer, through two symmetrical, all-His tetracoordination sites, revealing a classical His-Zn binding mode for the protein. Furthermore, the presence of a (Zn)₂-cacodylate complex in our second crystal form induces ligand swapping within the canonical His₄ zinc binding motif, thereby creating two new Zn-sites, one of which involves residues from symmetry-related molecules. Finally, we describe the calcium-induced S100A8 tetramer and reveal how zinc stabilizes this tetramer by tightening the dimer-dimer interface.

Conclusions

Our structures of Zn²⁺/Ca²⁺-bound hS100A8 demonstrate that S100A8 is a genuine His-Zn S100 protein. Furthermore, they show how zinc stabilizes S100A8 tetramerization and potentially mediates the formation of novel interdimer interactions. We propose that these zinc-mediated interactions may serve as a basis for the generation of larger oligomers in vivo.

     

The C-terminal domain of TPX2 is made of alpha-helical tandem repeats

Background

TPX2 (Targeting Protein for Xklp2) is essential for spindle assembly, activation of the mitotic kinase Aurora A and for triggering microtubule nucleation. Homologs of TPX2 in Chordata and plants were previously identified. Currently, proteins of the TPX2 family have little structural information and only small parts are covered by defined protein domains.

Methods

We have used computational sequence analyses and structural predictions of proteins of the TPX2 family, supported with Circular Dichroism (CD) measurements.

Results

Here, we report our finding that the C-terminal domain of TPX2, which is responsible of its microtubule nucleation capacity and is conserved in all members of the family, is actually formed by tandem repeats, covering well above 2/3 of the protein. We propose that this region forms a flexible solenoid involved in protein-protein interactions. Structural prediction and molecular modeling, combined with Circular Dichroism (CD) measurements reveal a predominant alpha-helical content. Furthermore, we identify full length homologs in fungi and shorter homologs with a different domain organization in diptera (including a paralogous expansion in Drosophila).

Conclusions

Our results, represent the first computational and biophysical analysis of the TPX2 proteins family and help understand the structure and evolution of this conserved protein family to direct future structural studies.

     

Comparison of human and mouse E-selectin binding to Sialyl-Lewis<Superscript>x</Superscript>

Background

During inflammation, leukocytes are captured by the selectin family of adhesion receptors lining blood vessels to facilitate exit from the bloodstream. E-selectin is upregulated on stimulated endothelial cells and binds to several ligands on the surface of leukocytes. Selectin:ligand interactions are mediated in part by the interaction between the lectin domain and Sialyl-Lewis x (sLe^x), a tetrasaccharide common to selectin ligands. There is a high degree of homology between selectins of various species: about 72 and 60 % in the lectin and EGF domains, respectively. In this study, molecular dynamics, docking, and steered molecular dynamics simulations were used to compare the binding and dissociation mechanisms of sLe^x with mouse and human E-selectin. First, a mouse E-selectin homology model was generated using the human E-selectin crystal structure as a template.

Results

Mouse E-selectin was found to have a greater interdomain angle, which has been previously shown to correlate with stronger binding among selectins. sLe^x was docked onto human and mouse E-selectin, and the mouse complex was found to have a higher free energy of binding and a lower dissociation constant, suggesting stronger binding. The mouse complex had higher flexibility in a few key residues. Finally, steered molecular dynamics was used to dissociate the complexes at force loading rates of 2000–5000 pm/ps². The mouse complex took longer to dissociate at every force loading rate and the difference was statistically significant at 3000 pm/ps². When sLe^x-coated microspheres were perfused through microtubes coated with human or mouse E-selectin, the particles rolled more slowly on mouse E-selectin.

Conclusions

Both molecular dynamics simulations and microsphere adhesion experiments show that mouse E-selectin protein binds more strongly to sialyl Lewis x ligand than human E-selectin. This difference was explained by a greater interdomain angle for mouse E-selectin, and greater flexibility in key residues. Future work could introduce similar amino acid substitutions into the human E-selectin sequence to further modulate adhesion behavior.

     

Case report of whole genome sequencing in the XY female: identification of a novel <Emphasis Type="Italic">SRY</Emphasis> mutation and revision of a misdiagnosis of androgen insensitivity syndrome

Background

The 46,XY female is characterised by a male karyotype and female phenotype arising due to any interruption in the sexual development pathways in utero. The cause is usually genetic and various genes are implicated.

Case presentation

Herein we describe a 46,XY woman who was first diagnosed with androgen insensitivity syndrome (testicular feminisation) at 18 years; however, this was later questioned due to the presence of intact Müllerian structures. The clinical phenotype suggested several susceptibility genes including SRY, DHH, NR5A1, NR0B1, AR, AMH, and AMHR2. To study candidate genes simultaneously, we performed whole genome sequencing. This revealed a novel and likely pathogenic missense variant (p.Arg130Pro, c.389G>C) in SRY, one of the major genes implicated in complete gonadal dysgenesis, hence securing this condition over androgen insensitivity syndrome as the cause of the patient’s disorder of sexual development.

Conclusion

This case highlights the emerging clinical utility of whole genome sequencing as a tool in differentiating disorders of sexual development.

     

A power analysis for future clinical trials on the potential adverse effects of SSRIs on amygdala reactivity

Treatment of adolescents with antidepressants may induce an increased risk for suicidality in this population. The activity of the amygdala during processing of emotional faces with functional Magnetic Resonance Imaging (fMRI) is a well-known measure of emotional dysregulation. Based upon data of our prematurely ended randomized clinical trial with fluoxetine (NTR3103) in anxious and or depressed girls (12–14 years of age) we calculated that with the found effect size of r = 0.66, compared to placebo, only 8 subjects are needed to demonstrate increased amygdala activity following 16 weeks of treatment with fluoxetine.     

Phosphodiesterase 4 inhibitors and drugs of abuse: current knowledge and therapeutic opportunities

Background

Long-term exposure to drugs of abuse causes an upregulation of the cAMP-signaling pathway in the nucleus accumbens and other forebrain regions, this common neuroadaptation is thought to underlie aspects of drug tolerance and dependence. Phosphodiesterase 4 (PDE4) is an enzyme that the selective hydrolyzes intracellular cAMP. It is expressed in several brain regions that regulate the reinforcing effects of drugs of abuse.

Objective

Here, we review the current knowledge about central nervous system (CNS) distribution of PDE4 isoforms and the effects of systemic and brain-region specific inhibition of PDE4 on behavioral models of drug addiction.

Methods

A systematic literature search was performed using the Pubmed.

Results

Using behavioral sensitization, conditioned place preference and drug self-administration as behavioral models, a large number of studies have shown that local or systemic administration of PDE4 inhibitors reduce drug intake and/or drug seeking for psychostimulants, alcohol, and opioids in rats or mice.

Conclusions

Preclinical studies suggest that PDE4 could be a therapeutic target for several classes of substance use disorder. We conclude by identifying opportunities for the development of subtype-selective PDE4 inhibitors that may reduce addiction liability and minimize the side effects that limit the clinical potential of non-selective PDE4 inhibitors. Several PDE4 inhibitors have been clinically approved for other diseases. There is a promising possibility to repurpose these PDE4 inhibitors for the treatment of drug addiction as they are safe and well-tolerated in patients.

     

Antibacterial effect of bestatin during periodontitis

Periodontitis is a polymicrobial disease inciting inflammatory destruction of the tooth-supporting tissues, i.e., periodontium. The initiation of this infectious disease is ascribed to the formation of subgingival biofilms. These biofilms cause stimulation of myriad of chronic inflammatory reactions by the affected tissue. The Gram-negative anaerobe Porphyromonas gingivalis is commonly found as part of the microbiota of subgingival biofilms, and is involved in the occurrence of the disease. P. gingivalis possesses numerous virulence factors supporting its survival, regulating its communication with other species in the biofilm, degrading host tissues. Fusobacterium nucleatum is pivotal for formation of biofilm and promotes growth and invasion properties of P. gingivalis. Bestatin is an aminopeptide inhibitor, produced by actinomycetes. It possesses antibacterial properties against P. gingivalis and F. nucleatum. The following review focuses on action of bestatin on the mentioned bacteria.