Modelling of Thyroid Peroxidase Reveals Insights into Its Enzyme Function and Autoantigenicity

Thyroid peroxidase (TPO) catalyses the biosynthesis of thyroid hormones and is a major autoantigen in Hashimoto’s disease—the most common organ-specific autoimmune disease. Epitope mapping studies have shown that the autoimmune response to TPO is directed mainly at two surface regions on the molecule: immunodominant regions A and B (IDR-A, and IDR-B). TPO has been a major target for structural studies for over 20 years; however, to date, the structure of TPO remains to be determined. We have used a molecular modelling approach to investigate plausible modes of TPO structure and dimer organisation. Sequence features of the C-terminus are consistent with a coiled-coil dimerization motif that most likely anchors the TPO dimer in the apical membrane of thyroid follicular cells. Two contrasting models of TPO were produced, differing in the orientation and exposure of their active sites relative to the membrane. Both models are equally plausible based upon the known enzymatic function of TPO. The “trans” model places IDR-B on the membrane-facing side of the myeloperoxidase (MPO)-like domain, potentially hindering access of autoantibodies, necessitating considerable conformational change, and perhaps even dissociation of the dimer into monomers. IDR-A spans MPO- and CCP-like domains and is relatively fragmented compared to IDR-B, therefore most likely requiring domain rearrangements in order to coalesce into one compact epitope. Less epitope fragmentation and higher solvent accessibility of the “cis” model favours it slightly over the “trans” model. Here, IDR-B clusters towards the surface of the MPO-like domain facing the thyroid follicular lumen preventing steric hindrance of autoantibodies. However, conformational rearrangements may still be necessary to allow full engagement with autoantibodies, with IDR-B on both models being close to the dimer interface. Taken together, the modelling highlights the need to consider the oligomeric state of TPO, its conformational properties, and its proximity to the membrane, when interpreting epitope-mapping data.     

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes.     

When None of Us Perform Better than All of Us Together: The Role of Analogical Decision Rules in Groups

During social interactions, groups develop collective competencies that (ideally) should assist groups to outperform average standalone individual members (weak cognitive synergy) or the best performing member in the group (strong cognitive synergy). In two experimental studies we manipulate the type of decision rule used in group decision-making (identify the best vs. collaborative), and the way in which the decision rules are induced (direct vs. analogical) and we test the effect of these two manipulations on the emergence of strong and weak cognitive synergy. Our most important results indicate that an analogically induced decision rule (imitate-the-successful heuristic) in which groups have to identify the best member and build on his/her performance (take-the-best heuristic) is the most conducive for strong cognitive synergy. Our studies bring evidence for the role of analogy-making in groups as well as the role of fast-and-frugal heuristics for group decision-making.     

Diversity and ecology of algae from the Nahal Qishon river,northern Israel

In 59 samples of periphyton and phytoplankton collected in 2002 - 2003 from the Nahal Qishon (Qishon River), northern Israel, we found 178 species from seven divisions of algae and cyanoprocaryotes. Diatoms, clorophytes, and cyanoprocaryotes prevail. Nitzschia and Navicula (Bacillariophyta) are the most abundant. Most of the species are cosmopolitan or widespread, except Lagynion janei (Chrysophyta), which is endemic for the Mediterranean Realm. About 17% of species (26) are new for Israel and five of them represent the first recorded genera: Crinalium endophyticum Crow, Actinocyclus normanii (Gregory) Hustedt, Rhizoclonium hieroglyphicum (Agardh) Kütz (Chlorophyta), Lagynion janei Bourelly, and Stylococcus aureus Chodat. Most of them come from a rare riverine assemblage with red alga Audouinella pygmea, as well as from the estuarine assemblage. Alkaliphiles predominate among the indicators of acidity, with few acidophiles confined to the communities under the impact of industrial wastes. Among the indicators of salinity, most numerous are the oligohalobien-indifferents and species adapted to a moderate salinity level. The relative species richness of ecological groups and the indices of saprobity are correlated with changes in conductivity, pH, and N-nitrate concentration. Indicators of organic pollution fall in the range of betameso- to alfamesosaprobic self-purification grades. Our studies show ecological significance of the Nahal Qishon as a model for a strongly disturbed aquatic ecosystem in the coastal zone of eastern Mediterranean.     

Structural and biochemical characterisation of a NAD+-dependent alcohol dehydrogenase from Oenococcus oeni as a new model molecule for industrial biotechnology applications

Alcohol dehydrogenases are highly diverse enzymes catalysing the interconversion of alcohols and aldehydes or ketones. Due to their versatile specificities, these biocatalysts are of great interest for industrial applications. The adh3-gene encoding a group III alcohol dehydrogenase was isolated from the gram-positive bacterium Oenococcus oeni and was characterised after expression in the heterologous host Escherichia coli. Adh3 has been identified by genome BLASTP analyses using the amino acid sequence of 1,3-propanediol dehydrogenase DhaT from Klebsiella pneumoniae and group III alcohol dehydrogenases with known activity towards 1,3-propanediol as target sequences. The recombinant protein was purified in a two-step column chromatography approach. Crystal structure determination and biochemical characterisation confirmed that Adh3 forms a Ni²⁺-containing homodimer in its active form. Adh3 catalyses the interconversion of ethanol and its corresponding aldehyde acetaldyhyde and is also capable of using other alcoholic compounds as substrates, such as 1,3-propanediol, 1,2-propanediol and 1-propanol. In the presence of Ni²⁺, activity increases towards 1,3-propanediol and 1,2-propanediol. Adh3 is strictly dependent on NAD⁺/NADH, whereas no activity has been observed with NADP⁺/NADPH as co-factor. The enzyme exhibits a specific activity of 1.1 U/mg using EtOH as substrate with an optimal pH value of 9.0 for ethanol oxidation and 8.0 for aldehyde reduction. Moreover, Adh3 exhibits tolerance to several metal ions and organic solvents, but is completely inhibited in the presence of Zn²⁺. The present study demonstrates that O. oeni is a group III alcohol dehydrogenase with versatile substrate specificity, including Ni²⁺-dependent activity towards 1,3-propanediol.     

Hofmeister ions control protein dynamics

Background

Recently, we have elaborated a thermodynamic theory that could coherently interpret the diverse effects of Hofmeister ions on proteins, based on a single physical parameter, the protein–water interfacial tension (Dér et al., Journal of Physical Chemistry B. 2007, 111, 5344–5350). This theory, implying a “liquid drop model”, predicts changes in protein conformational fluctuations upon addition of Hofmeister salts (containing either kosmotropic or chaotropic anions) to the medium.

Methods

Here, we report experimental tests of this prediction using a complex approach by applying methods especially suited for the detection of protein fluctuation changes (neutron scattering, micro-calorimetry, and Fourier-transform infrared spectroscopy).

Results

It is demonstrated that Hofmeister salts, via setting the hydrophobic/hydrophilic properties of the protein–water interface, control conformational fluctuations even in the interior of the typical membrane transport protein bacteriorhodopsin, around its temperature-induced, unusual α(II) → α(I) conformational transition between 60 and 90 °C. We found that below this transition kosmotropic (COOCH₃⁻), while above it chaotropic (ClO₄⁻) anions increase structural fluctuations of bR. It was also shown that, in each case, an onset of enhanced equilibrium fluctuations presages this phase transition in the course of the thermotropic response of bR.

Conclusions

These results are in full agreement with the theory, and demonstrate that predictions based on protein–water interfacial tension changes can describe Hofmeister effects and interpret protein dynamics phenomena even in unusual cases.

General significance

This approach is expected to provide a useful guide to understand the principles governing the interplay between protein interfacial properties and conformational dynamics, in general.     

Amelioration of age‐related brain function decline by Bruton's tyrosine kinase inhibition

One of the hallmarks of aging is the progressive accumulation of senescent cells in organisms, which has been proposed to be a contributing factor to age‐dependent organ dysfunction. We recently reported that Bruton's tyrosine kinase (BTK) is an upstream component of the p53 responses to DNA damage. BTK binds to and phosphorylates p53 and MDM2, which results in increased p53 activity. Consistent with this, blocking BTK impairs p53‐induced senescence. This suggests that sustained BTK inhibition could have an effect on organismal aging by reducing the presence of senescent cells in tissues. Here, we show that ibrutinib, a clinically approved covalent inhibitor of BTK, prolonged the maximum lifespan of a Zmpste24^?/? progeroid mice, which also showed a reduction in general age‐related fitness loss. Importantly, we found that certain brain functions were preserved, as seen by reduced anxiety‐like behaviour and better long‐term spatial memory. This was concomitant to a decrease in the expression of specific markers of senescence in the brain, which confirms a lower accumulation of senescent cells after BTK inhibition. Our data show that blocking BTK has a modest increase in lifespan in Zmpste24^?/? mice and protects them from a decline in brain performance. This suggests that specific inhibitors could be used in humans to treat progeroid syndromes and prevent the age‐related degeneration of organs such as the brain.