Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase

eEF2K [eEF2 (eukaryotic elongation factor 2) kinase] phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily, but instead belongs to a small group of so-called α-kinases. The activity of eEF2K is normally dependent upon Ca2+ and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. In the present study we have identified several autophosphorylation sites, including Thr348, Thr353, Ser366 and Ser445, all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser78, a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser78, Thr348 and Ser366 to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr348 was detected by immunoblotting after transfecting wild-type eEF2K into HEK (human embryonic kidney)-293 cells, but not after transfection with a kinase-inactive construct, confirming that this is indeed a site of autophosphorylation. Thr348 appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other α-kinases is also autophosphorylated and contributes to the activation of these enzymes [Crawley, Gharaei, Ye, Yang, Raveh, London, Schueler-Furman, Jia and Cote (2011) J. Biol. Chem. 286, 2607–2616]. Ser366 phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is phosphorylated not only autocatalytically but also in trans by other kinases

Belgio

Reactive oxygen species (ROS) are mainly produced in the mitochondrial (Larosa and Remacle, 2018) and in the photosynthetic electron transport chains (Pospíšil, 2009). Historically, ROS were only considered as toxic molecules for cells, leading to oxidation of proteins, lipids and DNA. Nowadays, the ROS-molecule H2O2 is increasingly being recognized as a signaling molecule due to the fact that it is relatively stable compared to the other ROS-molecules and H2O2 can potentially travel across membranes (Mittler, 2017). H2O2 signals via rapid reactions with protein cysteine sulfurs, which results in an altered protein structure and function (Pedre et al., 2018). Such cysteine modifications are known as S-sulfenylations (-SOH). So far, hundreds of sulfenylated proteins have been identified in the model plant Arabidopsis thaliana (Waszczak et al., 2014; Akter et al., 2015). In this project we want to (i) identify C. reinhardtii crucial redox enzymes which effect the phenotype under H2O2-stress inducing conditions; (ii) trap and identify sulfenylated proteins involved in the redox signaling, using dimedone-based carbon nucleophiles and mass spectrometry; (iii) in vitro characterize the oxidation kinetics and the oxidation induced structural changes on one of the identified redox-sensing proteins. This work is supported by the Fonds Wetenschappelijk Onderzoek – Vlaanderen (FWO) and the Fonds de la Recherche Scientifique – FNRS under EOS Project No. 30829584. REFERENCES 1.Akter S. et al., (2015) DYn-2 Based identification of Arabidopsis sulfenomes. Mol. Cell Proteomics 14:1183–1200 2.Larosa V. and Remacle C. (2018) Insight the respiratory chain and oxidative stress. Bioscience Rep. 38 BSR20171492 3.Mittler R. (2017) ROS are Good. Trends Plant Sci. 22:11-19 4.Pedre B. et al., (2018) Structural snapshots of OxyR reveal the peroxidatic mechanism of H2O2 sensing. Proc. Natl. Acad. Sci. 115:E11623-E11632 5.Pospíšil P. (2009) Production of reactive oxygen species by photosystem II as a response to light and temperature stress. Biochim Biophys Acta 10:1151-1160 6.Waszczak C. et al., (2014) Sulfenome mining in Arabidopsis thaliana. Proc. Natl. Acad. Sci. 111:11545-1155

Divide-and-Conquer Matrisome Protein (DC-MaP) Strategy: An MS-Friendly Approach to Proteomic Matrisome Characterization

Currently, the extracellular matrix (ECM) is considered a pivotal complex meshwork of macromolecules playing a plethora of biomolecular functions in health and disease beyond its commonly known mechanical role. Only by unraveling its composition can we leverage related tissue engineering and pharmacological efforts. Nevertheless, its unbiased proteomic identification still encounters some limitations mainly due to partial ECM enrichment by precipitation, sequential fractionation using unfriendly-mass spectrometry (MS) detergents, and resuspension with harsh reagents that need to be entirely removed prior to analysis. These methods can be technically challenging and labor-intensive, which affects the reproducibility of ECM identification and induces protein loss. Here, we present a simple new method applicable to tissue fragments of 10 mg and more. The technique has been validated on human ovarian tissue and involves a standardized procedure for sample processing with an MS-compatible detergent and combined centrifugation. This two-step protocol eliminates the need for laborious sample clarification and divides our samples into 2 fractions, soluble and insoluble, successively enriched with matrisome-associated (ECM-interacting) and core matrisome (structural ECM) proteins

Standardised workflow for mass spectrometry-based single-cell proteomics data processing and analysis using the scp package

Mass spectrometry (MS) based single-cell proteomics (SCP) explores cellular heterogeneity by focusing on the functional effectors of the cells - proteins. However, extracting meaningful biological information from MS data is far from trivial, especially with single cells. Currently, data analysis workflows are substantially different from one research team to another. Moreover,it is difficult to evaluate pipelines as ground truths are missing. Our team has developed the R/Bioconductor package called scp to provide a standardised framework for SCP data analysis. It relies on the widely used QFeatures and SingleCellExperiment data structures. In addition, we used a design containing cell lines mixed in known proportions to generate controlled variability for data analysis benchmarking. In this work, we provide a flexible data analysis protocol for SCP data using the `scp` package together with comprehensive explanations at each step of the processing. Our main steps are quality control on the feature and cell level, aggregation of the raw data into peptides and proteins, normalisation and batch correction. We validate our workflow using our ground truth data set. We illustrate how to use this modular, standardised framework and highlight some crucial steps

Generation of Organized Porcine Testicular Organoids in Solubilized Hydrogels from Decellularized Extracellular Matrix

Cryopreservation of immature testicular tissue (ITT) prior to chemo/radiotherapy is now ethically accepted and is currently the only way to preserve fertility of prepubertal boys about to undergo cancer therapies. So far, three-dimensional culture of testicular cells isolated from prepubertal human testicular tissue was neither efficient nor reproducible to obtain mature spermatozoa, and ITT transplantation is not a safe option when there is a risk of cancer cell contamination of the testis. Hence, generation of testicular organoids (TOs) after cell selection is a novel strategy aimed at restoring fertility in these patients. Here, we created TOs using hydrogels developed from decellularized porcine ITT and compared cell numbers, organization and function to TOs generated in collagen only hydrogel. Organotypic culture of porcine ITT was used as a control. Rheological and mass spectrometry analyses of both hydrogels highlighted differences in terms of extracellular matrix stiffness and composition, respectively. Sertoli cells (SCs) and germ cells (GCs) assembled into seminiferous tubule-like structures delimited by a basement membrane while Leydig cells (LCs) and peritubular cells localized outside. TOs were maintained for 45 days in culture and secreted stem cell factor and testosterone demonstrating functionality of SCs and LCs, respectively. In both TOs GC numbers decreased and SC numbers increased. However, LC numbers decreased significantly in the collagen hydrogel TOs (p < 0.05) suggesting a better preservation of growth factors within TOs developed from decellularized ITT and thus a better potential to restore the reproductive capacity

Optimizing the trapping and identification of sulfenylated proteins in the green microalga Chlamydomonas reinhardtii

In photosynthetic organisms, chloroplast is one of the main sources of reactive oxygen species (ROS). The most common ROS produced during photosynthesis are superoxide anion (O2-), singlet oxygen (1O2) and hydrogen peroxide (H2O2) [1]. In this study we mainly focused on H2O2 which can also act as a signalling molecule by oxidizing cysteine residues of proteins into their sulfenylated form. The aim of the project is to identify these proteins in order to establish their role during oxidative stress in the green microalga C. reinhardtii. We optimized a protocol for trapping sulfenylated proteins from [2] by using a benzothiazine-based chemoselective probe (BTD) [3] specific to recognize -SOH. We succeeded to label sulfenylated proteins after addition of H2O2. However, we are still in the process to optimize the workflow for modified peptides identification by mass spectrometry analyses. Preliminary results on wild type cells have shown that some photosynthetic proteins are susceptible to H2O2 modification, such us the light-harvesting complexes (LHC) of photosysthem II, LHCII3, LHCB3 and LHCII-1.3 important to capture light during photosynthesis. Our analyses will provide information on the Chlamydomonas sulfenome, and will be compared to the other post translational modifications already identified on the Chlamydomonas proteome, such as glutathionylation and nitrosylation. Funded by FNRS-FWO EOS Project 30829584 [1] Asada (2006). Plant Physiol. 141 (2): 391-396 [2] Huang et al. (2019). PNAS. 116 (42): 21256-21261 [3] Gupta et al. (2017) J. Am. Chem. Soc. 139 (159): 5588–559

AMPK activation by SC4 inhibits noradrenaline-induced lipolysis and insulin-stimulated lipogenesis in white adipose tissue.

The effects of small-molecule AMP-activated protein kinase (AMPK) activators in rat epididymal adipocytes were compared. SC4 was the most effective and submaximal doses of SC4 and 5-amino-4-imidazolecarboxamide (AICA) riboside were combined to study the effects of AMPK activation in white adipose tissue (WAT). Incubation of rat adipocytes with SC4 + AICA riboside inhibited noradrenaline-induced lipolysis and decreased hormone-sensitive lipase (HSL) Ser563 phosphorylation, without affecting HSL Ser565 phosphorylation. Preincubation of fat pads from wild-type (WT) mice with SC4 + AICA riboside inhibited insulin-stimulated lipogenesis from glucose or acetate and these effects were lost in AMPKα1 knockout (KO) mice, indicating AMPKα1 dependency. Moreover, in fat pads from acetyl-CoA carboxylase (ACC)1/2 S79A/S212A double knockin versus WT mice, the effect of SC4 + AICA riboside to inhibit insulin-stimulated lipogenesis from acetate was lost, pinpointing ACC as the main AMPK target. Treatment with SC4 + AICA riboside decreased insulin-stimulated glucose uptake, an effect that was still observed in fat pads from AMPKα1 KO versus WT mice, suggesting the effect was partly AMPKα1-independent. SC4 + AICA riboside treatment had no effect on the insulin-induced increase in palmitate esterification nor on sn-glycerol-3-phosphate-O-acyltransferase activity. Therefore in WAT, AMPK activation inhibits noradrenaline-induced lipolysis and suppresses insulin-stimulated lipogenesis primarily by inactivating ACC and by inhibiting glucose uptake

Thiol-disulphide independent in-cell trapping for the identification of peroxiredoxin 2 interactors

Hydrogen peroxide (H2O2) acts as a signalling molecule by oxidising cysteine thiols in proteins. Recent evidence has established a role for cytosolic peroxiredoxins in transmitting H2O2-based oxidation to a multitude of target proteins. Moreover, it is becoming clear that peroxiredoxins fulfil their function in organised microdomains, where not all interactors are covalently bound. However, most studies aimed at identifying peroxiredoxin interactors were based on methods that only detect covalently linked partners. Here, we explore the applicability of two thiol-disulphide independent in-cell trapping methodological approaches in combination with mass spectrometry for the identification of interaction partners of peroxiredoxin 2 (Prdx2). The first is biotin-dependent proximity-labelling (BioID) with a biotin ligase A (BirA*)-fused Prdx2, which has never been applied on redox-active proteins. The second is crosslinker co-immunoprecipitation with an N-terminally His-tagged Prdx2. During the initial characterisation of the tagged Prdx2 constructs, we found that the His-tag, but not BirA*, compromises the peroxidase and signalling activities of Prdx2. Further, the Prdx2 interactors identified with each approach showed little overlap. We therefore concluded that BioID is a more reliable method than crosslinker co-immunoprecipitation. After a stringent mass spec data filtering, BioID identified 13 interactors under elevated H2O2 conditions, including subunit five of the COP9 signalosome complex (CSN5). The Prdx2:CSN5 interaction was further confirmed in a proximity ligation assay. Taken together, our results demonstrate that BioID can be used as a method for the identification of interactors of Prdxs, and that caution should be exercised when interpreting protein-protein interaction results using tagged Prdx