Is Mn-Bound Substrate Water Protonated in the S2 State of Photosystem II?

In spite of great progress in resolving the geometric structure of the water-splitting Mn4OxCa cluster in photosystem II, the binding sites and modes of the two substrate water molecules are still insufficiently characterized. While time-resolved membrane-inlet mass spectrometry measurements indicate that both substrate water molecules are bound to the oxygen-evolving complex (OEC) in the S2 and S3 states (Hendry and Wydrzynski in Biochemistry 41:13328–13334, 2002), it is not known (1) if they are both Mn-bound, (2) if they are terminal or bridging ligands, and (3) in what protonation state they are bound in the different oxidation states Si (i = 0, 1, 2, 3, 4) of the OEC. By employing 17O hyperfine sublevel correlation (HYSCORE) spectroscopy we recently demonstrated that in the S2 state there is only one (type of) Mn-bound oxygen that is water exchangeable. We therefore tentatively identified this oxygen as one substrate ‘water’ molecule, and on the basis of the finding that it has a hyperfine interaction of about 10 MHz with the electron spin of the Mn4OxCa cluster, we suggest that it is bound as a Mn–O–Mn bridge within a bis-μ2 oxo-bridged unit (Su et al. in J Am Chem Soc 130:786–787, 2008). Employing pulse electron paramagnetic resonance, 1H/2H Mims electron-nuclear double resonance and 2H-HYSCORE spectroscopies together with 1H/2H-exchange here, we test this hypothesis by probing the protonation state of this exchangeable oxygen. We conclude that this oxygen is fully deprotonated. This result is discussed in the light of earlier reports in the literature

Species Used for Drug Testing Reveal Different Inhibition Susceptibility for 17beta-Hydroxysteroid Dehydrogenase Type 1

Steroid-related cancers can be treated by inhibitors of steroid metabolism. In searching for new inhibitors of human 17beta-hydroxysteroid dehydrogenase type 1 (17β-HSD 1) for the treatment of breast cancer or endometriosis, novel substances based on 15-substituted estrone were validated. We checked the specificity for different 17β-HSD types and species. Compounds were tested for specificity in vitro not only towards recombinant human 17β-HSD types 1, 2, 4, 5 and 7 but also against 17β-HSD 1 of several other species including marmoset, pig, mouse, and rat. The latter are used in the processes of pharmacophore screening. We present the quantification of inhibitor preferences between human and animal models. Profound differences in the susceptibility to inhibition of steroid conversion among all 17β-HSDs analyzed were observed. Especially, the rodent 17β-HSDs 1 were significantly less sensitive to inhibition compared to the human ortholog, while the most similar inhibition pattern to the human 17β-HSD 1 was obtained with the marmoset enzyme. Molecular docking experiments predicted estrone as the most potent inhibitor. The best performing compound in enzymatic assays was also highly ranked by docking scoring for the human enzyme. However, species-specific prediction of inhibitor performance by molecular docking was not possible. We show that experiments with good candidate compounds would out-select them in the rodent model during preclinical optimization steps. Potentially active human-relevant drugs, therefore, would no longer be further developed. Activity and efficacy screens in heterologous species systems must be evaluated with caution

Electron Transfer from Cyt b559 and Tyrosine-D to the S2 and S3 states of the water oxidizing complex in Photosystem II at Cryogenic Temperatures

The Mn4CaO5 cluster of photosystem II (PSII) catalyzes the oxidation of water to molecular oxygen through the light-driven redox S-cycle. The water oxidizing complex (WOC) forms a triad with Tyrosine(Z) and P-680, which mediates electrons from water towards the acceptor side of PSII. Under certain conditions two other redox-active components, Tyrosine(D) (Y-D) and Cytochrome b (559) (Cyt b (559)) can also interact with the S-states. In the present work we investigate the electron transfer from Cyt b (559) and Y-D to the S-2 and S-3 states at 195 K. First, Y-D (aEuro cent) and Cyt b (559) were chemically reduced. The S-2 and S-3 states were then achieved by application of one or two laser flashes, respectively, on samples stabilized in the S-1 state. EPR signals of the WOC (the S-2-state multiline signal, ML-S-2), Y-D (aEuro cent) and oxidized Cyt b (559) were simultaneously detected during a prolonged dark incubation at 195 K. During 163 days of incubation a large fraction of the S-2 population decayed to S-1 in the S-2 samples by following a single exponential decay. Differently, S-3 samples showed an initial increase in the ML-S-2 intensity (due to S-3 to S-2 conversion) and a subsequent slow decay due to S-2 to S-1 conversion. In both cases, only a minor oxidation of Y-D was observed. In contrast, the signal intensity of the oxidized Cyt b (559) showed a two-fold increase in both the S-2 and S-3 samples. The electron donation from Cyt b (559) was much more efficient to the S-2 state than to the S-3 state

X-ray absorption spectroscopy

This review gives a brief description of the theory and application of X-ray absorption spectroscopy, both X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS), especially, pertaining to photosynthesis. The advantages and limitations of the methods are discussed. Recent advances in extended EXAFS and polarized EXAFS using oriented membranes and single crystals are explained. Developments in theory in understanding the XANES spectra are described. The application of X-ray absorption spectroscopy to the study of the Mn4Ca cluster in Photosystem II is presented

Making New "New AI" Friends : Designing a Social Robot for Diabetic Children from an Embodied AI Perspective

Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.Robin is a cognitively and motivationally autonomous affective robot toddler with "robot diabetes" that we have developed to support perceived self-efficacy and emotional wellbeing in children with diabetes by providing them with positive mastery experiences of diabetes management in a playful but realistic and natural interaction context. Underlying the design of Robin is an "Embodied" (formerly also known as "New") Artificial Intelligence approach to robotics. In this paper we discuss the rationale behind the design of Robin to meet the needs of our intended end users (both children and medical staff), and how "New AI" provides a suitable approach to developing a friendly companion that fulfills the therapeutic and affective requirements of our end users beyond other approaches commonly used in assistive robotics and child-robot interaction. Finally, we discuss how our approach permitted our robot to interact with and provide suitable experiences of diabetes management to children with very different social interaction styles.Peer reviewedFinal Published versio

Exome sequencing of pleuropulmonary blastoma reveals frequent biallelic loss of TP53 and two hits in DICER1 resulting in retention of 5p-derived miRNA hairpin loop sequences

Pleuropulmonary blastoma is a rare childhood malignancy of lung mesenchymal cells that can remain dormant as epithelial cysts or progress to high-grade sarcoma. Predisposing germline loss-of-function DICER1 variants have been described. We sought to uncover additional contributors through whole exome sequencing of 15 tumor/normal pairs, followed by targeted resequencing, miRNA analysis and immunohistochemical analysis of additional tumors. In addition to frequent biallelic loss of TP53 and mutations of NRAS or BRAF in some cases, each case had compound disruption of DICER1: a germline (12 cases) or somatic (3 cases) loss-of-function variant plus a somatic missense mutation in the RNase IIIb domain. 5p-Derived microRNA (miRNA) transcripts retained abnormal precursor miRNA loop sequences normally removed by DICER1. This work both defines a genetic interaction landscape with DICER1 mutation and provides evidence for alteration in miRNA transcripts as a consequence of DICER1 disruption in cancer

Importance of Post-Translational Modifications for Functionality of a Chloroplast-Localized Carbonic Anhydrase (CAH1) in Arabidopsis thaliana

Background: The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells. Methodology/Principal Findings: Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited. Conclusions/Significance: We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.This work was supported by the Swedish Research Council (VR), the Kempe Foundations and Carl Tryggers Foundation to GS, and grant numbers BIO2006-08946 and BIO2009-11340 from the Spanish Ministerio de Ciencia e Innovación (MICINN) to A

Insights in 17β-HSD1 Enzyme Kinetics and Ligand Binding by Dynamic Motion Investigation

BACKGROUND: Bisubstrate enzymes, such as 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), exist in solution as an ensemble of conformations. 17beta-HSD1 catalyzes the last step of the biosynthesis of estradiol and, thus, it is a potentially attractive target for breast cancer treatment. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the conformational transitions of its catalytic cycle, a structural analysis of all available crystal structures was performed and representative conformations were assigned to each step of the putative kinetic mechanism. To cover most of the conformational space, all-atom molecular dynamic simulations were performed using the four crystallographic structures best describing apoform, opened, occluded and closed state of 17beta-HSD1 as starting structures. With three of them, binary and ternary complexes were built with NADPH and NADPH-estrone, respectively, while two were investigated as apoform. Free energy calculations were performed in order to judge more accurately which of the MD complexes describes a specific kinetic step. CONCLUSIONS/SIGNIFICANCE: Remarkably, the analysis of the eight long range trajectories resulting from this multi-trajectory/-complex approach revealed an essential role played by the backbone and side chain motions, especially of the betaF alphaG'-loop, in cofactor and substrate binding. Thus, a selected-fit mechanism is suggested for 17beta-HSD1, where ligand-binding induced concerted motions of the FG-segment and the C-terminal part guide the enzyme along its preferred catalytic pathway. Overall, we could assign different enzyme conformations to the five steps of the random bi-bi kinetic cycle of 17beta-HSD1 and we could postulate a preferred pathway for it. This study lays the basis for more-targeted biochemical studies on 17beta-HSD1, as well as for the design of specific inhibitors of this enzyme. Moreover, it provides a useful guideline for other enzymes, also characterized by a rigid core and a flexible region directing their catalysis

Impact of primary kidney disease on the effects of empagliflozin in patients with chronic kidney disease: secondary analyses of the EMPA-KIDNEY trial

Background: The EMPA KIDNEY trial showed that empagliflozin reduced the risk of the primary composite outcome of kidney disease progression or cardiovascular death in patients with chronic kidney disease mainly through slowing progression. We aimed to assess how effects of empagliflozin might differ by primary kidney disease across its broad population. Methods: EMPA-KIDNEY, a randomised, controlled, phase 3 trial, was conducted at 241 centres in eight countries (Canada, China, Germany, Italy, Japan, Malaysia, the UK, and the USA). Patients were eligible if their estimated glomerular filtration rate (eGFR) was 20 to less than 45 mL/min per 1·73 m2, or 45 to less than 90 mL/min per 1·73 m2 with a urinary albumin-to-creatinine ratio (uACR) of 200 mg/g or higher at screening. They were randomly assigned (1:1) to 10 mg oral empagliflozin once daily or matching placebo. Effects on kidney disease progression (defined as a sustained ≥40% eGFR decline from randomisation, end-stage kidney disease, a sustained eGFR below 10 mL/min per 1·73 m2, or death from kidney failure) were assessed using prespecified Cox models, and eGFR slope analyses used shared parameter models. Subgroup comparisons were performed by including relevant interaction terms in models. EMPA-KIDNEY is registered with ClinicalTrials.gov, NCT03594110. Findings: Between May 15, 2019, and April 16, 2021, 6609 participants were randomly assigned and followed up for a median of 2·0 years (IQR 1·5–2·4). Prespecified subgroupings by primary kidney disease included 2057 (31·1%) participants with diabetic kidney disease, 1669 (25·3%) with glomerular disease, 1445 (21·9%) with hypertensive or renovascular disease, and 1438 (21·8%) with other or unknown causes. Kidney disease progression occurred in 384 (11·6%) of 3304 patients in the empagliflozin group and 504 (15·2%) of 3305 patients in the placebo group (hazard ratio 0·71 [95% CI 0·62–0·81]), with no evidence that the relative effect size varied significantly by primary kidney disease (pheterogeneity=0·62). The between-group difference in chronic eGFR slopes (ie, from 2 months to final follow-up) was 1·37 mL/min per 1·73 m2 per year (95% CI 1·16–1·59), representing a 50% (42–58) reduction in the rate of chronic eGFR decline. This relative effect of empagliflozin on chronic eGFR slope was similar in analyses by different primary kidney diseases, including in explorations by type of glomerular disease and diabetes (p values for heterogeneity all >0·1). Interpretation: In a broad range of patients with chronic kidney disease at risk of progression, including a wide range of non-diabetic causes of chronic kidney disease, empagliflozin reduced risk of kidney disease progression. Relative effect sizes were broadly similar irrespective of the cause of primary kidney disease, suggesting that SGLT2 inhibitors should be part of a standard of care to minimise risk of kidney failure in chronic kidney disease. Funding: Boehringer Ingelheim, Eli Lilly, and UK Medical Research Council