A large ‘discovery’ experiment: Gender Initiative for Excellence (Genie) at Chalmers University of Technology

Sweden tops gender equality rankings, but Swedish academia is still lacking women in top positions. To address gender inequality in its faculty, Chalmers University of Technology has invested 300 million SEK (30 million Euros) over 10\ua0years in Gender initiative for Excellence (Genie). Genie aims to increase the university’s success and excellence via gender equality efforts. In this editorial, we want to share insights on explicit efforts during Genie’s first 2.5\ua0years with the goal to inspire and advise other universities and researchers

Structural analyses of immune cell receptor signalling and activation

Structural biology is a scientific field where the aim is to observe macromolecules on a atomic level to understand their functions. Often these macromolecules are proteins that make, almost, everything happen within the cells; from hormones and enzymes to building blocks and controlling gene expression. With structural biology tools, scientists can visualize structures, interactions and mobility within the proteins to get an insight in the chain of events in a cell. In this thesis, the function of signalling has been in focus. Both signalling within the immune system through a membrane bound receptor that finds an invading pathogen and signals into the cells “alert, we have an invader” where the immune system reacts. How the signalling passes from the outside of the cell to the inside is still not revealed. One part of this thesis investigates the outside of an immune cell and the other, the inside, past the membrane. Proteins are chains of amino acids that are predestined to either fold into a stable structure or stay loose and flexible. Superantigens are very stable proteins; they are toxins, made to last and conquer. The opposite are the intracellular flexible domains of the immune receptors which belong to a class of proteins, so-called intrinsically disordered proteins, IDPs, which are less investigated but omnipresent. In this thesis some flexible domains of the immune receptors have efficiently been produced in a cell free protein synthesis and examined by NMR, using a new setup of acquisition and analysis. All domains are lacking secondary structure and a well-defined three-dimensional structure. The proteins investigated more in depth within the work of this thesis, show tendency for α-helical regions, most likely of functional significance. Viruses are evolved to use its host and get a free ride. Here we explore the interaction of one SIV (orthologous to HIV) protein with one of the intracellular flexible domains of the T-cell receptor, which leads to down regulation of the receptor resulting in immune deficiency. This interaction is unique in that no changes in the very sensitive NMR spectra are seen; yet other techniques indicate specific interaction. As the SIV protein is abusing the immune system, superantigens hijack the immune system by crosslinking the T-cell receptor to an antigen-presenting cell displaying pieces of an invading pathogen on it´s surface, and by this start an extreme immune response, sometimes lethal. This superantigen can circumvent the intricate, specific and effective immune system and they are up to date thought to interact with the β-chain of the T-cell receptor. We show in this thesis by structural biology techniques such as x-ray and NMR that a superantigen interacts with the α-chain of the TCR. This is the first structure structurally determined ternary complex of an antigen-MHC-superantigen-TCR, a paradigm shift in superantigen biology

The intrinsically disordered cytoplasmic domain of the T cell receptor zeta chain binds to the nef protein of simian immunodeficiency virus without a disorder-to-order transition.

Intrinsically disordered proteins are thought to undergo coupled binding and folding upon interaction with their folded partners. In this study, we investigate whether binding of the intrinsically disordered T cell receptor zeta cytoplasmic tail to the well-folded simian immunodeficiency virus Nef core domain is accompanied by a disorder-to-order transition. We show that zeta forms a 1:1 complex with Nef and remains unfolded in the complex. Thus, our findings oppose the generally accepted view of the behavior of intrinsically disordered proteins and provide new evidence of the existence of specific interactions for unfolded protein molecules

Rational Discovery of T Helper Epitopes Specific for Bovine Infections with Mycobacterium avium ssp paratuberculosis

Superantigens (SAgs) are bacterial toxins that interact with immunoreceptors, T cell receptor (TCR) and major histocompatibility complex (MHC) class II, conventionally through the variable beta-domain of TCR (TCRV beta). They induce a massive release of cytokines, which can lead to diseases such as food poisoning and toxic shock syndrome. In this study, we report the X-ray structure of the ternary complex between staphylococcal enterotoxin H (SEH) and its human receptors, MHC class II and TCR. The structure demonstrates that SEH predominantly interacts with the variable alpha-domain of TCR (TCRV alpha), which is supported by nuclear magnetic resonance (NMR) analyses. Furthermore, there is no contact between MHC and TCR upon complex formation. Structural analyses suggest that the major contact points to TCRV alpha are conserved among other bacterial SAgs. Consequently, a new dimension of SAg biology emerges, suggesting that in addition to the conventional interactions with the TCRV beta domain, SAgs can also activate T cells through the TCRV alpha domain

AMPK and AKT protein kinases hierarchically phosphorylate the N-terminus of the FOXO1 transcription factor, modulating interactions with 14-3-3 proteins

Forkhead box protein O1 (FOXO1) is a transcription factor involved in various cellular processes such as glucose metabolism, development, stress resistance, and tumor suppression. FOXO1's transcriptional activity is controlled by different environmental cues through a myriad of posttranslational modifications. In response to growth factors, the serine/threonine kinase AKT phosphorylates Thr$^{24}$ and Ser$^{256}$ in FOXO1 to stimulate binding of 14-3-3 proteins, causing FOXO1 inactivation. In contrast, low nutrient and energy levels induce FOXO1 activity. AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis, partly mediates this effect through phosphorylation of Ser$^{383}$ and Thr$^{649}$ in FOXO1. In this study, we identified Ser$^{22}$ as an additional AMPK phosphorylation site in FOXO1's N terminus, with Ser$^{22}$ phosphorylation preventing binding of 14-3-3 proteins. The crystal structure of a FOXO1 peptide in complex with 14-3-3 σ at 2.3 Å resolution revealed that this is a consequence of both steric hindrance and electrostatic repulsion. Furthermore, we found that AMPK-mediated Ser$^{22}$phosphorylation impairs Thr$^{24}$ phosphorylation by AKT in a hierarchical manner. Thus, numerous mechanisms maintain FOXO1 activity via AMPK signaling. AMPK-mediated Ser$^{22}$ phosphorylation directly and indirectly averts binding of 14-3-3 proteins, whereas phosphorylation of Ser$^{383}$ and Thr$^{649}$ complementarily stimulates FOXO1 activity. Our results shed light on a mechanism that integrates inputs from both AMPK and AKT signaling pathways in a small motif to fine-tune FOXO1 transcriptional activity

Highly efficient NMR assignment of intrinsically disordered proteins: application to B- and T cell receptor domains.

We present an integrated approach for efficient characterization of intrinsically disordered proteins. Batch cell-free expression, fast data acquisition, automated analysis, and statistical validation with data resampling have been combined for achieving cost-effective protein expression, and rapid automated backbone assignment. The new methodology is applied for characterization of five cytosolic domains from T- and B-cell receptors in solution

AMPK and AKT protein kinases hierarchically phosphorylate the N-terminus of the FOXO1 transcription factor, modulating interactions with 14-3-3 proteins

Forkhead box protein O1 (FOXO1) is a transcription factor involved in various cellular processes such as glucose metabolism, development, stress resistance, and tumor suppression. FOXO1\u27s transcriptional activity is controlled by different environmental cues through a myriad of posttranslational modifications. In response to growth factors, the serine/threonine kinase AKT phosphorylates Thr24 and Ser256 in FOXO1 to stimulate binding of 14-3-3 proteins, causingFOXO1inactivation. In contrast, low nutrient and energy levels induce FOXO1 activity. AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis, partly mediates this effect through phosphorylation of Ser383 and Thr649 in FOXO1. In this study, we identified Ser22 as an additional AMPK phosphorylation site in FOXO1\u27s N terminus, with Ser22 phosphorylation preventing binding of 14-3-3 proteins. The crystal structure of a FOXO1 peptide in complex with 14-3-3 σ at 2.3 \uc5 resolution revealed that this is a consequence of both steric hindrance and electrostatic repulsion. Furthermore, we found that AMPK-mediated Ser22 phosphorylation impairs Thr24 phosphorylation by AKT in a hierarchical manner. Thus, numerous mechanisms maintain FOXO1 activity via AMPK signaling. AMPK-mediated Ser22 phosphorylation directly and indirectly averts binding of 14-3-3 proteins, whereas phosphorylation of Ser383 and Thr649 complementarily stimulates FOXO1 activity. Our results shed light on a mechanism that integrates inputs from both AMPK and AKT signaling pathways in a small motif to fine-tune FOXO1 transcriptional activity

Progress of targeted acquisition versus total measurement time for a 120 µM sample of CD79a.

<p>Build-ups are shown for the number of assigned residues and the number of detected peaks in individual BEST-TROSY-type experiments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062947#pone.0062947-Favier1" target="_blank">[14]</a>.</p

Zoomed region of the ¹⁵N-HSQC of CD79a in different conditions.

<p>(<b>A</b>) NaPi buffer, (<b>B</b>) 6 M urea, (<b>C</b>) 20% TFE, (<b>D</b>) reduced spin label (MTSL) attached to CD79a, (<b>E</b>) K4C/C35S form, (<b>F</b>) Y25E/Y36E form. Selected peaks are annotated to show rearrangements of the signal position for the different conditions (19Y, 35M) or position of the specific mutations (4K, 33C, 25Y, 36Y). Peaks outside the zoomed region are shown as arrows pointing towards the correct position.</p