Characterization of cancer related Orai1 mutants A122T, N147S and P164H / submitted by Sascha Berlansky BSc

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JOHANNES KEPLER UNIVERSITÄT LINZ Altenberger Straße 69 4040 Linz, Österreich jku.at DVR 0093696 Submitted by Sascha Berlansky BSc Submitted at JKU Institute of Biophysics Department: Ion Channels Supervisor Assoc. Univ.-Prof. Dr. Christoph Romanin Co-Supervisor Dr. Irene Frischauf, MLBT May 2020

Characterization of

cancer related Orai1

mutants A122T, N147S

and P164H

Master thesis

To obtain the academic degree of

Master of Science

In the Master’s program

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ACKNOWLEDGEMENTS

First of all, I would like to thank A. Univ.-Prof. Dr. Christoph Romanin for giving me the opportunity to work in his team.

I am very grateful that my co-supervisor Dr. Irene Frischauf supported me throughout my time in this group with her knowledge. Thank you for taking the time to answer all my questions! This thesis would have never been written without your support.

I would also like to thank the entire Ion-Channel group for their friendly and patient support and all the practical advice I got from them. Special thanks to Dr. Marc Fahrner, Dr. Victoria Lunz, Ing. Sabine Buchegger, Adéla Tiffner MSc. and Sonja Lindinger MSc. for helping me with all those minor questions, which accumulate during a normal working day in the lab.

Finally, I would like to acknowledge that I am very fortunate to have Matthias Sallinger BSc. and Herwig Grabmayr MSc. at my side, not just coworkers, but also friends. Thank you for all the years of support, motivation and the fun we had together!

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EIDESSTATTLICHE ERKLÄRUNG

Ich erkläre an Eides statt, dass ich die vorliegende Masterarbeit selbstständig und ohne fremde Hilfe verfasst, andere als die angegebenen Quellen und Hilfsmittel nicht benutzt bzw. die wörtlich oder sinngemäß entnommenen Stellen als solche kenntlich gemacht habe.

Die vorliegende Masterarbeit ist mit dem elektronisch übermittelten Textdokument identisch.

Linz, 21.05.2020 Ort, Datum

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Abstract

The role of Ca2+ as a signalling molecule is essential to fundamental cellular functions such as proliferation, cell growth, gene expression or the secretion of bioactive molecules. Intracellular Ca2+ homeostasis is maintained via regulatory systems such as store operated Ca2+ channels (SOCC), which are the key players of store-operated Ca2+_{entry (SOCE).}

Orai1 proteins build hexameric Ca2+ selective channels in the plasma membrane. Upon activation by STIM1, which acts as a Ca2+_{sensor protein in the ER, the Ca}2+_{release activated}

Ca2+_{(CRAC) channels allow the influx of Ca}2+_{ions into the cell, replenishing the intracellular}

Ca2+_{store within the ER.}

Throughout years of research several disease-related STIM1 and Orai1 mutations have been identified. This thesis focuses on three cancer associated Orai1 mutants, taken from the cBioPortal database. The mutants A122T (malignant mixed Mullerian tumours of the uterus), N147S (germinal center B-cell type cancer) and P164H (lung adenocarcinoma) were investigated for their impact on pore structure at pore lining residues R91, F99 and E106 by utilizing a cysteine cross-linking approach. Furthermore, the electrophysiological channel characteristics were measured via whole cell patch clamp recordings, protein localization was verified by fluorescence microscopy and a NFAT screen was carried out to measure possible modulations of gene transcription downstream the Ca2+_{signalling cascade.}

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Kurzfassung

Die Rolle von Kalzium in der zellulären Signalübertragung ist fundamental für grundlegende Prozesse von Zellen, beispielsweise in der Proliferation, dem Zellwachstum, der Genexpression oder der Sekretion von bioaktiven Molekülen. Intrazelluläre Kalzium -Homöostase wird durch Systeme wie den speichergesteuerten Kalzium Kanal (store operated Ca2+ channels - SOCC) reguliert, welche Hauptakteure im speichergesteuerten Kalzium-Einlass (store-operated Ca2+

entry - SOCE) von Zellen sind.

Orai1 Proteine sind hexamere Kalzium-selektive Ionenkanäle in der Plasmamembran. Nach ihrer Aktivierung, welche durch STIM-Proteine, die als Kalziumsensoren in der Membran des endoplasmatischen Retikulum dienen, wird der Einstrom von Kalzium-Ionen aus dem extrazellulären Raum und somit das Auffüllen der intrazellulären Kalzium-Speicher im ER ermöglicht.

Durch jahrelange Forschung sind mehrere krankheitsassoziierte Mutationen von STIM1 und Orai1 identifiziert worden. In der folgenden Arbeit werden drei krebsassoziierte Orai1 Mutanten aus der Datenbank von cBioPortal untersucht. Die Mutanten A122T (Müllerscher Mischtumor), N147S (diffuses großzelliges B-Zelllymphom) und P164H (Adenokarzinom der Lunge) wurden auf strukturelle Änderungen der Kanalpore an den Positionen R91, F99 and E106, welche dem Inneren der Pore zugewandt sind, durch Cystein-Crosslinking untersucht. Zusätzlich wurden elektrophysiologische Kanalcharakteristiken mittels whole cell patch clamp gemessen, die Lokalisation der Proteine mit Fluoreszenzmikroskopie überprüft, sowie NFAT-Screening zur Überprüfung von möglichen Auswirkungen auf die Transkription von Genen durch Änderungen in der Signalkaskade durchgeführt.

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1. Introduction

1.1. Calcium in cellular life

1.1.1. Calcium functions

Calcium (Ca2+_{) is a divalent cation with the atomic number 20. It is found in the second group of}

the periodic table, making it an alkaline earth metal fourth period of the periodic table of the elements alongside the other alkaline earth metals Beryllium (Be), Magnesium (Mg), Strontium (Sr), Barium (Ba), and Radium (Ra). [1]

Ca2+ also plays an important role in cellular life. It is necessary for essential cellular processes such as cell-differentiation, proliferation as well as apoptosis and several transcription factors (e.g. NFAT) are sensitive to changes in Ca2+ concentrations via the phosphatase Calcineurin. Ca2+ _{also plays a critical role for the release of neurotransmitters into the synaptic cleft by}

triggering vesicle fusion of neurotransmitter containing vesicles with the synaptic membrane via SNARE-protein complexes. [2]

Besides the already mentioned functions, Ca2+ _{is also responsible for the}_{regulation of muscle}

contractions in smooth muscle tissue as well as skeletal muscle cells. [3] It has essential functions in the immune system and is required for the activation of T-cells by inducing the interaction between them and antigen presenting cells (APCs). [4] [5]

Ca2+ is found in very low concentrations in the intracellular space at approximately 100 nM and in an around 20,000-fold higher concentration in the mM range extracellularly. [6]

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1.1.1. Ca2+ binding motifs

In order to fulfill its biological functions, Ca2+ _{often binds to proteins that contain calcium binding}

motifs. Two of the best known motifs are the EF hand helix-loop-helix motif and the annexin Ca2+-binding motif. The annexin protein family binds Ca2+ via four repeat domains, each containing a calcium binding site (CBS) consisting of negatively charged glutamate and aspartate regions. [7] [8]

Kretsinger et al labelled the binding motif „EF hand“ due to the structural similarity of the alpha helices to a human hand (see Figure 2). The EF hand motif consists of a pair of alpha helical domains that are connected by a loop of usually 12 amino acids. Five of these twelve amino acids contain negatively charged oxygen atoms, which help calcium binding. The remaining residues typically form a hydrophobic core that binds and stabilizes the two helices in their conformation. [9]

Figure 2. Left: Structure of the EF hand. The colored helices correspond to the fingers of the

symbolic hand. Upon binding of the Ca2+ ion, the thumb helix moves to the open conformation. [9] Right: 3D-representation of the molecular Ca2+_{-binding geometry of an EF hand. [9]}

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1.1.2. Ca2+ homeostasis

Mg2+_{and K}+_{are the main cationic ions, which are found in a relative high concentration in the}

intracellular space, whereas Ca2+ and Na+ act as their extracellular counterparts. The concentration of Ca2+_{in the extracellular space of animals ranges between values from 1 to 10}

mM. In the cytosol, the concentration is much lower and only 100 nM of Ca2+_{usually is}

measured. [8]

This enormous 10,000-fold concentration gradient for Ca2+_{across the cell membrane causes a}

strong driving force for Ca2+ influx. Measurements show that cells under resting conditions have a low membrane permeability to Ca2+_{but a small increase in permeability leads to large Ca}2+

influx. This is achieved via Ca2+_{permeable ion channels in the plasma membrane. Various Ca}2+

permeable channels exist: voltage-operated channels (VOC), second messenger-operated channels (SMOC), store-operated channels (SOC), receptor-operated channels (ROC) and the antiporter system Na+_-Ca2+_{exchanger (NCX). [10] An overview of some of the major regulators}

of Ca2+ _{is shown in Figure 3.}

Figure 3. Depiction of some of the major regulators of calcium homeostasis. Ca2+_{-Pumps and}

Ca2+-channels (PMCA, NCX, and SOCE) regulate in- and out-flux the cytosol. G-protein– coupled receptors initiate signals that modify calcium stores downstream via signalling molecules such as IP3. Calcium ATPases SERCA or SPCA1 localized on organelles such as the

golgi apparatus monitor and replenish intracellular storage sites. Sensing and replenishment of the ER/SR via Stim1 and ORAI1 are discussed in further chapters. [11]

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In order to keep Ca2+_{homeostasis at physiological range, cells do not rely on Ca}2+ _channels

alone, in which the influx of Ca2+ ions follows their electrochemical gradient. Cells need to build up and maintain the concentration of Ca2+ _{ions on both sides of the plasma membrane in order}

to function. This is achieved by ATP-driven Ca2+_{-pumps called „Ca}2+_{ATPases“. There are three}

known types of Ca2+_{ATPases in vertebrates: plasma membrane Ca}2+_{ATPases (PMCAs),}

sarco/endoplasmic reticulum Ca2+_{ATPases (SERCAs), and secretory pathway Ca}2+_ATPases

(SPCAs). [8]

Figure 4. Reaction mechanism of Ca2+_{-ATPases. In a simplified model, two functional states}

exist: E1 and E2. In the E1 state, in which the ATP is hydrolyzed, the pumps have high Ca2+

affinity while in the E2 state the affinity becomes much lower which leads to the release of Ca2+

at the extracellular side of the membrane. [12]

There are several inhibitors for different classes of ATPases, e.g. the Lanthanum ion La3+ or orthovanadate, which tend to inhibit all ATPases. La3+_{inhibitions the ATPases by mimicking the}

divalent cationic charge of calcium, but it blocks the channel due to a higher atomic radius of rLanthanum = 195 pm (compared to rCalcium= 180 pm). Orthovanadate acts as a competitive inhibitor

of ATPases, alkaline and acid phosphatases, and protein-phosphotyrosine phosphatases. [12] The specific inhibition of SERCAs triggered by thapsigargin (TG), which is ineffective against the other ATPases, is implemented in the NFAT-measurement experiments used for this thesis. Another way to artificially deplete Ca2+_{stores of cells is via chelating the ions with suitable}

compounds such as glycol diamine tetraacetic acid (EGTA), which was used in patch clamp measurements.

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1.1.3. Role of Ca2+ in cell cycle regulation

The cell cycle consists of four phases: first gap phase (G1), the DNA-synthesis phase (S), the second gap phase (G2) and the mitosis phase (M). In order to ensure the proper division of the cells, there are control mechanisms termed “cell cycle checkpoints”. During these checkpoints, cells either continue with the next phase, initiate DNA repair mechanisms or apoptosis. If errors occur during these cell cycle checkpoints, cells that should initiate apoptosis due to irreparable damaged DNA or failed DNA repair mechanisms could continue with their mitotic division, leading to pathogenic conditions such as carcinogenesis.

Essential regulators of the cell cycle are cyclins and cyclin-dependent kinases (CDKs). The expression as well as the activity of these regulators is dependent on Ca2+_{. [13]}

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1.2. Store operated calcium entry

1.2.1. SOCE

The concept of store-operated calcium entry, a process in which the release of stored calcium leads to the opening of plasma membrane calcium channels, has its roots in the late 1970’s, and was formalized in 1986 by Putney. [14]

It was discovered that upon the depletion off intracellular Ca2+_{stores of the ER, Ca}2+_influx

occurred via the plasma membrane, resulting in replenishment of intracellular Ca2+_stores.

This pathway was termed „store-operated Ca2+_{entry“ (SOCE) in which a reduction of}

intracellular store concentration of Ca2+_{leads to Ca}2+_{influx over the plasma membrane via}

store-operated Ca2+_{channels (SOCCs). [10]}

The investigation of these Ca2+_{entry pathways was done via electrophysiological techniques in}

mast cells, followed by the discovery, that SOCE takes place in nearly all cell types. [15] [16] [17] In 1992 Hoth & Penner termed these channels „Ca2+_{release-activated Ca}2+_{(CRAC) channels“.}

[18]

The exact mechanisms and the unusual process that involves proteins from two different cellular compartments remained unclear in the early 1990s. CRAC channels were known to exhibit very specific properties that distinguished them from other known Ca2+_{channels. These properties}

include a high Ca2+_{selectivity with a permeability ratio of 1:1000 of Ca}2+_{to Na, a low single}

channel conductance with approximately 20fs as well as many known Ca2+_{-dependent feedback}

mechanisms. [10]

In 2005 Liou et al. identified the stromal interaction molecule 1 (STIM1) as a Ca2+_sensing

protein in the membrane of the ER. [19] One year later Orai proteins, first termed “CRACM1”, were discovered by Feske et al. as the cause for severe combined immune deficiency via RNAi-approaches and genetic linkage analysis [20]

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1.1.1. STIM

The stromal interaction molecule (STIM) is a single-pass transmembrane protein located in the ER membrane that regulates store operated calcium entry (SOCE) via its function as a Ca2+

sensor. It senses a drop in ER Ca2+ concentration through its EF hand domain that binds to Ca2+ ions in resting state. [21] [22] [23]

The

N-terminus that contains a Ca2+_{binding EF-hand motif as}

well as a sterile alpha motif (SAM) is located in the ER lumen (Figure 6). The interaction of the EF-hand originates from an aspartate and glutamate rich helix-loop-helix motif. [24] [25] [26] The C-terminus of STIM1 remains in the cytosol. It contains three coiled-coil regions (CC1, CC2, CC3), a serine-proline- as well as a lysine-rich region and the CRAC modulatory domain (CAD). While the N-terminus of STIM1 is crucial for of Ca2+_{sensing, the C-terminus is essential for}

STIM1 oligomerization and its interaction with Orai1. Essential domains for Orai1 activation are OASF (Orai activating STIM fragment), CAD (CRAC activating domain), SOAR (STIM-Orai activating region), Ccb9 (Figure 7). [21, 27]

Figure 6. Depiction of hypothetical resting state of STIM1. ER lumen displayed in dark green

(bottom), cytosol displayed in lighter green (top). Ca2+_{is bound to the EF-Hand. [21]}

Figure 7. Depiction of linearized STIM1, displaying essential regions. ER lumen displayed in

dark green (left), cytosol displayed in lighter green (right). EF-Hand and SAM as well as CC3 and CC2 are shown as 3D models. [21]

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1.1.2. Orai

Orai proteins are tetra-spanning membrane proteins that form highly selective Ca2+_{-channels in}

the plasma membrane (Figure 8 and Figure 10). Their gating mechanism depends on the coupling to STIM1 proteins, which leads to an open state that allows the influx of Ca2+ ions into the cell. [28] [21] In mammals three types of highly conserved Orai proteins exist (Orai 1-3) which show tissue-specific expression levels. All three Orai proteins share an identical TM1 helix that forms the inner pore, surrounded by the other transmembrane helices. (Figure 10) The position E106 in TM1 was shown to be important for the selectivity for Ca2+_{ions. Mutating a wild}

type glutamate to aspartate or glutamine leads to a vast alteration in ion selectivity of the channel. [29] A mutation to aspartate (E106D) seems to increase the diameter of pore, leading to a higher permeability for larger cations like Na+, Ba2+, and Sr2+. Exchanging the glutamate with an uncharged amino acid leads to a non-functional channel. [30] Position R91 in TM1 seems to function as an electrostatic gate. Mutations to hydrophilic residues allowed normal functioning of the channel whereas hydrophobic mutations such as the SCID mutant (R91W) lead to non-functional channels. [31, 32] Orai proteins have a mass of approximately 30 kDa and their C- and N-termini are both located in the cytoplasm. Two extracellular and one intracellular loop connect the four transmembrane helices of Orai. [28] A CRAC channel is formed by six Orai1 subunits, enabling the possibility of potential homo- and heteromeric channels. [33] All members of the Orai family contain a coiled-coil domain in their C-terminus that is essential for the interaction with STIM1. Likewise, the N-terminus, which is located close to the C-terminus in the cytoplasm, is important for the interaction with STIM1 and both termini are necessary for channel activation. [34] [35]

It is assumed that the assembly of CRAC channels by multimerisation of Orai subunits is realized by interaction of the transmembrane regions, since the deletion of the C-termini doesn’t affect channel assembly. [34] [36] Biochemical and fluorescence studies provided evidence for a tetrameric assembly model of the CRAC channel. However, the crystal structure of the

Drosophila Orai protein revealed a hexameric assembly. [37] Although the crystal structure with

its 3.35 Å resolution provides the best representation of an Orai structure, it should be stated that the mutant used in this experiment lacks parts of the C- and N-termini as well as the TM1-TM2 and the TM1-TM2-TM3 loops and contains mutations at positions C224S, P276R, P277R and C283T.

In dOrai six TM1 helices form the pore that allows the Ca2+ ions to pass. The other transmembrane helices form the outer layers of the pore and offer structural support of the channel. [24] TM3 and TM4 are shown to be the most divergent domains between Orai isoforms. [21] [38] [39] Residues within TM3 also have been shown to modulate the gating and permeation even though they are not directly inside the pore. The current knowledge allows for a division of CRAC channel units into for parts: 1) a negatively charged selectivity filter composed of glutamates, 2) a hydrophobic region with three α-helical turns, 3) a basic domain that is suggested to act as an anion-coordinator (R91, K87, R83) and 4) a cytosolic domain containing two α-helical turns with a length of approximately 20 Å called “extended TM1 Orai1 N-terminal (ETON) region” that functions as a binding partner for STIM1. [21] [40]

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Figure 8. Left: Cartoon side view of an Orai1 monomer displaying N- and C-termini as well as all

4 transmembrane domains in different colours. Right: top view of an Orai hexamer, displaying a channel pore width of approximately 6 Å [21]

Figure 9. Cartoon representing a cross-section through the

Orai1 pore, displaying pore-lining TM1 helices and the corresponding amino acids.

Negatively charged amino acids are coloured in cyan, hydophobic amino acids in green and positive amino acids are shown in red. [21]

Figure 10. Approximate positions of A122 (red),

N147 (green) and P164 (blue) are shown in a cartoon of a linearized Orai1 (top) as well as a membrane-embedded Orai1 cartoon model (left). Adapted from [21]

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1.1.3. STIM/Orai interaction

The coupling of the cytosolic strand of STIM1 to Orai1 domains induces a conformational change in the CRAC channel, opening the gate and thereby leading to Ca2+ influx into the cell. The interaction of Orai1 with STIM1 includes the cytosolic region of STIM1 as well as the C-terminus and the N-C-terminus of Orai1, also located in the cytosol. [21] As long as Ca2+_{in the ER}

is abundant, it is bound to the EF-hand, keeping STIM1 in a resting state, and the STIM1 proteins remain uniformly distributed in the ER membrane. Upon store depletion of the ER the EF-hand senses a decrease in Ca2+ concentration and STIM1 alternates to its extended state. [28] The activated STIM1 proteins redistribute and oligomerize at ER-PM junctions, leading to the formation of STIM1 puncta. Orai1 proteins accumulate on the opposite side of the ER-PM junctions in the plasma membrane. One hypothesis to explain this process is a diffusion trap in which activated STIM1 diffusing in the ER becomes trapped at junctions through interactions with the plasma membrane, and STIM1 then traps Orai1 through binding of its calcium release-activated calcium activation domain, leading to an release-activated Orai1 (Figure 11). [41]

Figure 11. CRAC activation model. STIM1 is shown at resting state (left), transitioning to an

active state, and finally activating Orai1 (right), leading to an influx of Ca+2_{into the cytoplasm.}

[42]

During STIM1-Orai1 coupling of the Orai1 C-terminus and the coiled-coil 2 domain (CC2) of STIM1 involve mainly hydrophobic and ionic interactions. All members of the Orai family include a highly conserved terminal region (aa72-90) termed “extended transmembrane Orai1 N-terminal region” (ETON) that corresponds to the cytosolic TM1-extension. Mutants that lack the ETON (Δ1-76) or the whole N-terminus lose their function. [21] [40]

Both, the N- and C-termini of Orai1, are necessary for proper coupling to STIM1 and it has been suggested that STIM1 first binds to the Orai1 C-terminus before interacting with the N-terminus. [21] A Stim1 CC2 dimer binds to the C-termini of Orai1 and forms the hydrophobic and basic STIM-Orai association pocket (SOAP).

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Upon Stim1-Orai1-coupling, the channel opens and allows Ca2+_{influx. Prakriya et al. had}

proposed a pore rotation model for channel opening, in which the torsion of the TM1 helix is linked with conformational changes in the Orai1 C-terminus after coupling to STIM1. In this model the hydrophobic side chains of pore-lining residues V102 and F99, which create a barrier for Ca2+_{permeation in the closed state rotate approximately 20° outwards to open the channel.}

[43] [44]

In contrast, Dong et al. propose a „twist-to-open“ gating mechanism. By MD-simulations based on the dOrai structure they show that a coupling of TM1 to TM3 (R83-E149 and K85-E173) seems to be crucial for the activation of Orai channels and that a series of motions leads to channel opening. [45]

Frischauf et al have shown a small local widening of the pore (~1-2 Å) occurs during the opening of the channel, suggesting two gates in both the hydrophobic and basic region of the pore. Gain-of-function H134A mutants were shown to create hydrogen bonds between amino acid side chains that are facing the channels pore, such as S90 and R91. Additionally, this mutation decreases the hydrophobic gating barriers by creating a chain of water molecules through the channels pore. [46]

Long et al. crystallized the open dOrai channel by taking advantage of the H134A Orai1 gain-of-function mutation. [47] The open crystal suggests conformational changes and straightening of the TM4 and the extended TM4 region (M4ext) upon channel opening. [24]

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1.3. Calcium SOCE and cancer

1.3.1. STIM, Orai and cancer

Accumulating evidence suggests that SOCE plays a critical role in cancer cell proliferation, metastasis as well as tumor vascularization and antitumor immunity. [48] [49] [49] [48]. Ca2+

signalling via SOCE leads to different effects depending on the tumor stage. At tumor initiation stage, it induces genetic changes in premalignant cells. This can cause malignant transformations in cell, e.g. SOCE is responsible for the secretion of vascular endothelial growth factor (VEGF), which promotes the proliferation of endothelial cells.

Different tumor tissues show overexpression of Orai1 and STIM1 as well as different mutations of Orai1 and STIM1 proteins. Targeting SOCE has significant potential as anticancer treatment. Pharmacological inhibition of Orai1 and STIM1 can diminish the growth of colorectal, breast, liver, melanoma and clear cell renal cancer cells. A knockdown of STIM1 was shown to support chemotherapy-induced apoptosis in pancreatic as well as lung cancer cells. It has been shown that Orai3 is involved in the tumorigenesis of estrogen receptor-positive breast cancer. [50] During angiogenesis, one of the hallmarks of cancer, the vascular endothelial growth factor (VEGF) and the corresponding VEGF-receptors induce Ca2+_{store depletion, thereby inducing}

SOCE in human endothelial cells during angiogenesis. A knockdown of STIM or Orai1 was able to minimize VEGF-induced Ca2+_{influx in human umbilical vein endothelial cells (HUVECs),}

leading to inhibition of cell proliferation and migration. [51] [50]

In this master thesis, three cancer related mutants (A122T, N147S and P164H) from the cBioPortal database are investigated (see task). A short summary of the related cancer types is provided in the following paragraphs.

1.3.1. A122T linked to malignant mixed Mullerian tumour of the uterus

A malignant mixed Mullerian tumour (MMMT), also termed “uterine carcinosarcoma”, is a rare type of tumor in the uterus, the ovaries, the fallopian tubes and other tissues that contains both carcinomatous (epithelial tissue) and sarcomatous (connective tissue) components. [52]

These tumours are a dedifferentiated or metaplastic form of endometrial carcinoma, displaying histological features of both endometrial carcinoma and sarcoma. [53] Cervical MMMT usually occur in post-menopausal women with mean age at diagnosis of 61 to 69 years. The commonest clinical features are vaginal bleeding, abnormal vaginal cytology and polypoidal cervical mass. [54] [55] [56]. MMMT account for between 2-5% of all tumors derived from the uterus. [57] The survival of MMMT patients is determined primarily by depth of invasion and stage (four stages are classified). MMMTs are highly malignant whereas a stage I tumor has an expected five-year survival rate of 50%, while the overall five-year survival rate is less than 20%. [58]

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1.3.2. N147S linked to germinal center B-cell type cancer

Germinal centers (GCs) are sites within the lymphatic system and the spleen, in which mature B cells proliferate, differentiate and mutate their antibody genes during an immune response to an infection. They develop after the activation of follicular B cells by T-dependent antigens. [59] Germinal Center B-Cell type cancer is one of the three subtypes of diffuse large B-cell lymphoma that is known besides activated B-cell-like lymphoma and primary mediastinal B-cell lymphoma. [60] Gene-expression profiling has been used to define 3 molecular subtypes of diffuse large B-cell lymphoma (DLBCL), termed germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, and primary mediastinal B-cell lymphoma (PMBL). The Germinal Center B-Cell Type seem to arise from normal germinal center B cells, whereas the activated B-cell-like type may arise from postgerminal center B cells that are arrested during plasmacytic differentiation, and PMBLs may arise from thymic B cells. [60] Usually germinal center B cells manifest many different hallmarks of cancer. Most of the B‐cell neoplasms originate from the GC reaction, and display many different point mutations, structural genomic lesions, and genetic and epigenetic clonal diversity. The dominant biological theme of germ cell derived lymphomas is mutation of genes, which are involved in epigenetic regulation mechanisms and immune receptor signalling that comes into play at critical transitional stages of the GC reaction. [61]

1.3.1. P164H linked to lung adenocarcinoma

Lung adenocarcinoma is the most common primary lung cancer which belongs to the non-small cell lung cancers (NSCLC) and has a strong association with previous smoking. However, it is also the most common subtype of lung cancer that is diagnosed amongst non-smokers. Adenocarcinoma of the lung usually evolves from the mucosal glands and represents about 40% of all lung cancers and remains the leading cause of cancer deaths. Lung adenocarcinoma usually occurs in the lung periphery, and in many cases, may be found in scars or areas of chronic inflammation. [62] Adenocarcinoma is mostly diagnosed at an advanced or metastatic stage, when patients show symptoms outside the respiratory tract, these extrapulmonary manifestations are termed „paraneoplastic syndromes”. One of the most common paraneoplastic syndromes associated with adenocarcinoma is hypercalcemia of malignancy. It is more common in squamous cell carcinoma but it can occur as well in adenocarcinomas. Cancer cells cause increased bone resorption by upregulating the activity of osteoclasts, leading to a breakdown of bone tissue followed by a release of Ca2+_{into the}

bloodstream. This effect is caused by tumor cells that release the parathyroid hormone-related peptide (PTHrP), which has an upregulating effect on osteoclasts. Symptoms of elevated Ca2+ levels in the bloodstream include fatigue, increased thirst, constipation, polyuria and nausea. [63]. Studies of adenocarcinoma tumour tissues showed that Orai3 is overexpressed in lung adenocarcinomas when compared to non-tumour tissues by 66.5% compared to 32% overexpression for Orai2 and 31% for Orai1. [64]

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1.4. Task

Three cancer-related Orai1 mutations (A122T, N147S and P164H) from the cBioPortal database which are linked to different cancer types (see chapter Orai1 and cancer) were examined with a combination of three different analytical techniques:

Western Blotting for the analysis of structural changes at important channel residues R91 and

F99 (both positions are supposed to act as a gate of the channel) as well as E106 (selectivity filter) via disulfide-crosslinking studies.

Whole-Cell Patch Clamp measurements upon store depletion caused by EGTA to analyse

changes in channel conductance caused by the mutations.

Fluorescence Microscopy to A) investigate the localisation behaviour of the mutants via

YFP-tags as well as B) analyse the effect of the mutations onto the Ca2+_{dependent nuclear}

transcription factor (NFAT)

In order to examine if the introduced mutations have an impact onto the Orai1 pore, they were investigated via cysteine-crosslinking. The cysteine-mutations allow the formation of disulfide-bonds via crosslinking of residues in close proximity, leading to the formation of hOrai1-dimers. Comparing the rate of dimerization between metated and wild type proteins hints towards a structural change of the given positions, namely if the mutation leads to altered proximity of the hOrai1 monomeres at the given cysteine positions. To prevent unwanted crosslinking with natural occurring cysteines in hOrai1, only cysteine-free hOrai1 constructs (Δ64, C126,143,195V, N223A) were used for this experiment. The mutation N223A leads to a non-glycosylated mutant.

The patch clamp data provides insights into alterations in the ion conductance of the channel such as information if the channel is still functional, ion influx is reduced or increased or if the channel is constitutively open.

Finally, the NFAT screen delivers information of a change in transcriptional activation due to altered Ca2+_{influx downstream in the Ca}2+_{signal cascade that might be caused by muations.}

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Figure 16: Depiction of the experimental workflow. Molecular biological preparation steps

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1.5. Research techniques

1.5.1. SDS-Page

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting are used to separate proteins according to their molecular mass. Due to the strength of the bands also a semi-quantification of proteins is possible through this technique. Proteins are denatured by SDS molecules which act as detergents. The SDS molecules are charged negatively and shield the natural charges of different amino acids, thereby allowing a constant mass-to-charge-ratio of the amino acids, which enables an electrophoretic separation according to size in an electrical field in a polyacrylamide gel. The polyacrylamide gel consists of acrylamide molecules which are polimerized via the addition of ammonium persulfate (APS) and N,N,N,N- tetramethylenediamine (TEMED) that act as oxidizing agents.

Different acrylamide concentrations determine the strength of the polymer-network, thereby modulating the pore size and separation strength of the PAGE-gel. The gel typically consists of two gels known as stacking and separation gel. The stacking gel is constituted of a lower acrylamide concentration and a more acidic pH. When a current is applied Cl- migrates faster towards the stacking gel due to their small size and their negative charge, whereas the SDS-protein complexes migrate slower due to their size. Glycine ions, which are uncharged in the stacking gel due to the lower pH migrate slow. However, when they reach the stacking gel, which has a higher pH, glycine gets deprotonated. This leads to an acceleration of the ions inside the gel that leads to sharper protein bands (termed “collection effect”). [65]

Figure 12. Schematic representation of a SDS-PAGE setup. Stacking gel is shown in green,

running gel is displayed in grey. The anode, which applies an attractive force to the negative charge SDS-protein complexes is placed at the bottom of the setup, the cathode is positioned on

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1.5.2. Western blot and immunodetection

Western blotting is usually used after SDS-PAGE to transfer the separated proteins onto a membrane (nitrocellulose or PVDF membrane) for detection purposes. The PVDF membrane is placed between a SDS-gel and a cathode. The negatively charged proteins migrate from the SDS-gel to the membrane via electrophoretic transfer. For detection of the proteins of interest, specific antibodies are used after free binding sides on the membrane are blocked (usually with BSA or non-fat dried milk powder). Detection takes place through an enzymatic reaction by antibody-labelled enzymes, such as horseradish peroxidase, which produce a luminescence signal that can be made visible on an X-ray film. [67]

Figure 13. Schematic representation of a western blot setup. On the left side the assembly of

the sandwich is shown, the right side displays the electrophoretic transfer. The membrane needs to be placed towards the anode and the gel towards the cathode for correct protein transport.

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1.5.3. Patch-clamp electrophysiology

The patch clamp measurements enable identification and classification of ion channels and their corresponding currents. In patch clamp the ion flow through one (single channels) or multiple channels (whole cell mode) is recorded after applying voltages. For this, a glass pipette tip with a diameter of approximately 1-5µm is attached to the membrane by approaching the tip to the cell membrane with a micromanipulator and forming the seal via a suction device. Changes in conductance (ions flowing through the channel) and kinetic properties of the cannel (opening/closing speed) of ion channels have been identified with this method. To record the data, two electrodes (usually Ag/AgCl electrodes) are necessary. One inside the glass tip and the second electrode is placed into the cell bath. The setup configuration for patch clamp measurements typically consists of a Faraday cage (isolation of the equipment from electrical noises), a vibration isolation table, one or several microscopes for cell imaging, micromanipulators to move and position the electrodes, low voltage amplifiers, a perfusion system (distribution of buffers and solutions) as well as a computer for data acquisition and stimulus generation. [68]

Figure 14. Schematic representation of different patch clamp recording setups.

a) The cell attached mode is realised by suction without breaking of the membrane, in a whole cell as configuration the membrane is broken to access the cytosol of the cell with the electrode

inside the pipette.

b) Measurement recordings of cell-attached and whole cell mode show. Outwardly rectifying single channel currents as well as currents resulting from two channels are shown at the cell attached mode of the left side. In the whole cell mode outwardly rectifying total currents are

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1.5.4. Confocal fluorescence microscopy

Fluorescence microscopy is a technique that combines fluorescence modifications of proteins with microscopically detection techniques to increase the contrast between a signal (cells, tissues) and the background of a sample. Proteins of interest are labelled with different fluorophores, which allows the imaging of specific locations or interactions between proteins. Fluorescence dyes have specific absorption and excitation wavelengths. Fluorescence molecules occur in their so-called ground state (S0), in which electrons are excited upon exposure of specific wavelengths. This leads to a shift (termed “Stoaks shift”) to a higher energy state of electrons consisting of vibrational and rotational states (excited states S1,S2). The energy needed to shift an electron form S0 to the excited state is inversely related to the photon’s wavelength (E=h*c/λ with h as Plank’s constant c speed of light and λ wavelength of light in vacuum). Depending on the excitation wavelength, electrons undergo shifts either in the S1 or higher orbital numbers. The shift back from the excited state to the ground state is achieved by vibrational relaxation and photons of specific wavelengths are emitted. Alexander Jablonski described this process in a diagram named termed “Jablonski diagram”. [70] Confocal fluorescence microscopy describes the combination of fluorescence microscopy with a confocal microscopy system. Via a confocal laser-scanning microscope (CLSM) the out-of-focus haze can be improved, leading to a higher contrast for thicker samples. A laser beam is focused to a small spot of sample, leading to excitation of fluorescent molecules. The resulting emission is further imaged through a pinhole that blocks light out of the focus. [71]

Figure 15. Schematic representation of a confocal and spinning disk microscope setup.

a) The excitation land emission pathways are focused at a specific point at the sample. Out of focus light is blocked via a pinhole. [72]

b) Representative configuration of a spinning disk microscope. The collector disk and a pinhole disk scan spots on the sample with a high framerate. Emitted light from the sample is guided via

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2. Methods

2.1. General information

Two different plasmid vectors were used: peYFP-C1 containing the hOrai1 sequence for the fluorescence studies and pcDNA 3.1/V5-His-TOPO containing Δ64 Cys-free (C126,143,195V-no glycosylation site N223A) hOrai1 mutants for crosslinking-studies.

PCR primers were designed using QuikChange® Primer Design Program by Agilent Technologies and manufactured by Eurofins Genomics oligonucleotide synthesis.

The DNA constructs are verified by sequencing by Eurofins Genomics. Sequencing results were translated into amino-acid sequences via ExPASy Translate Tool (SIB Swiss Institute of Bioinformatics), followed by alignments with the hOrai1 wildtype sequence for the validation of the constructs utilizing Clustal Omega Multiple Sequence Alignment Program (EMBL-EBI). Finished constructs were stored at -20°C. Plasmid concentrations for lipofection (BioRad) were determined with a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific).

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2.2. Plasmid construction

2.2.1. PCR

The following mutations were inserted into the vectors (Figure 17) via site directed mutagenesis by PCR in a FlexCycler (Analytik Jena AG):

Table 1: List of constructs

Figure 17: Illustration of the used vector systems. pcDNA3.1/V5-His-TOPO vector from

Invitrogen (left) and pEYFP-C1 vector from BD Bioscience Clontech (right).

Temperature Time Cycles

95°C 2 min 1 95°C 40 sec 18 60°C 40 sec 68°C 8 min 68°C 10 min 1 4°C --

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Substance Volume [µL]

Nuclease free H2O (Promega) 37.75

Pfu polymerase 10x buffer (Promega 10x) 5

Dimethyl sulfoxide 1.5

dNTP mix 10mM (Promega) 1.25

Forward primer 125ng (Eurofins Genomics) 1.25 Reverse primer 125ng (Eurofins Genomics) 1.25

10-100µg Template DNA 1

Pfu polymerase (Promega) 1

Total volume 50

Table 3. PCR-program components

Point mutation Primer sequence

A122T Fw. 5'-caggcactgaaggtgatgagcagcccc-3´ Rev. 5'-ggggctgctcatcaccttcagtgcctg-3' N147S Fw. 5'-caccgcctcgatgctgggcaggatgca-3' Rev. 5'-tgcatcctgcccagcatcgaggcggtg-3' P164H Fw. 5'-ggtcaaggagtcccaccatgagcgcatgc-3' Rev. 5'-gcatgcgctcatggtgggactccttgacc-3'

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2.2.2. DPN I digestion & PCR validation

To validate a successful PCR-reaction 15µl of the PCR samples were mixed with 3µl of loading dye (Orange 6x Loading dye Promega) and subjected to 1% agarose gel containing Midori Green Advance DNA/RNA stain (NIPPON Genetics Europe) for 20 minutes at 110V. The rest of the sample (35µl) and 1µl of the enzyme DPN I were incubated to ensure the degradation of the template plasmids that were methylated by E. coli and lack the desired mutations. A Lambda HindIII Marker (Promega) was used for size comparison. Figure 18 shows a representative gel electrophoreses with peYFP-C1 vectors containing hOrai1 mutants.

Figure 18. Depiction of the Lambda Hind III marker (left) [73], and gel

electrophoresis (right). The distance of the fragments corresponds to the 6557bp band of the marker. This indicates successful PCR. Δ64 Orai1 contains 237 amino acids, which corresponds to 711bp for the insert. The vector has 5,5kb, therefore vector + insert have 6211bp.

2.2.3. Transformation into competent E. coli

To amplify the PCR product, the plasmids were transferred into competent E. coli. The E. coli, which were stored at -80°C were thawn on ice for 15 minutes. Next, 3µl of the DNA samples were added to 50µl of the E.coli, followed by incubation for 20 minutes on ice, followed by a 2 minute heat shock at 42°C. After the heat shock 250µl of pre-warmed LB medium were added to the samples, followed by incubation at 37°C whilst shaking for 1 hour for regeneration. After incubation the samples were pipetted into petri dishes containing kanamycin-agar for the peYFP-C1 vectors and ampicillin-agar plates for the pcDNA3.1/V5-His-TOPO vectors and incubated for growth overnight at 37°C.

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2.2.4. Miniprep

After overnight incubation of the transformed E. coli single colonies were picked and added to eprouvettes containing 3mL of LB-Medium and the corresponding antibiotic (Kanamycin / Ampicillin). The eprouvettes were incubated whilst shaking at 37°c overnight.

To purify the plasmids the PureYieldTM Miniprep protocol [74] by Promega was with the following modifications:

Elution (Step 10) was carried out with 60µl instead of 30µl and nuclease free H2O instead of the

Promega Elution Buffer was used.

2.2.5. Sequencing

The Miniprep-DNA was sent off to Eurofins Genomics for sequencing. After receiving the sequence, it was verified if the desired mutations were successfully integrated into the plasmids by comparing the results to the wildtype. The wildtype Orai sequence was searched on PubMed (Accession number NM_032790.3), translated to amino acid code with Expasy Translate [75] and aligned by the clustal omega multiple sequence alignment tool. [76]

Figure 19. Representative sequence alignment of Δ64 hOrai1 (the first 63 amino acids are

deleted in the wildtype) with E106C (yellow), A122T (red), cysteine-free (green) and the non-glycosylation mutation N223A (purple)

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2.2.6. Midiprep

After validation of the mutated plasmids the PureYieldTM_{Midiprep protocol [77] was carried out to}

further amplify the plasmids for transfection. For this, sequence-validated E. coli form the eprouvettes were added into 50mL of LB-medium with the corresponding antibiotic in an 250mL Erlenmeyer flask and incubated overnight at 37°c whilst shaking.

The flasks containing the transformed E.coli were centrifuged on the next day for 15 minutes at 5000 rpm and the supernatant was discarded. Next, the PureYieldTM Midiprep Quick protocol was carried out with a modification at Step 6: centrifugation for 20 minutes at 8000 rpm.

A NanoDrop measurement was done to determine the plasmid-concentration of the samples and the appropriate volume for the transfection was calculated (10µg of DNA per petri dish of HEK293 cells).

2.2.7. Transfection

To transfer the plasmids that were obtained via Midiprep into HEK293 cells, lipofection with TransFectinTM_{(Biorad) was carried out. For this, 800µl of transfection-DMEM (without FCS &}

antibiotics), a volume containing 10µg of plasmid-DNA and 10µL of TransFectinTM were mixed and incubated for 20 minutes to form DNA-liposome-complexes. Next, the solution was carefully added to HEK293 cells in petri dishes without washing away the adherent HEK293 cells and incubated for 20-22 hours at 37°C. The same treatment was carried out for the mock-samples that were used as blank values for the hOrai1 expression of the HEK293 cells, however these samples were prepared without the addition of the plasmid-DNA.

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2.3. Western blot

2.3.1. Lysis and cysteine-crosslinking

To purify the proteins the following lysis protocol was carried out after cells were transfected and incubated overnight:

Protocol Protein Purification out of HEK293 cells:

The medium is discarded and the cells are rinsed off the bottom of the Petri dish with 1mL HBSS (Biowest ) +1mM EDTA buffer.

The solution is centrifuged for 1min at 4600rpm.

The supernatant is discarded and the pellet resuspended in 1mL HBSS+1mM EDTA buffer, followed by centrifugation for 1min at 4600rpm.

This washing step is then repeated.

After the centrifugation the pellet is resuspended in 500µL Lysis Buffer, mixed with 15µL Protease Inhibitor and incubated on ice for 15min.

Then the solution is pulled up and down a syringe (diameter 0,4x20mm, Braun) for 5 times. Next the solution is centrifuged for 10min at 4600rpm.

The supernatant is now stored at -20°C.

An aliquot of 21µL is treated with 5µL CuP-solution for 5min, then the reaction is stopped by addition of 5µL Quenching solution.

The sample was incubated with 10µL Laemmli buffer for 10min at 55°C before loading 21µL onto the SDS-gel.

The SDS-page is run at 150V for 70 min in a 12% gel (see 3.3.2).

To separate proteins from cell fragments, another centrifugation step for 10 minutes at 4600 rpm is performed. The supernatant that contains the hOrai1 proteins is transferred into fresh

Eppendorf tubes and stored at -20°C.

Lysis buffer

20mM Tris-HCL 0.788g

2mM EDTA 0.146g

100mM NaCl 1.461g

Glycerol 10% 25mL

Table 5. Lysis buffer components

Cu2+_{-phenanthroline solution} _{Quenching solution (pH 7.4)}

CuSO4 1mM TrisHCl 50mM

1,10-phenantroline 1.3mM N-Ethylmaleimide 20mM

NaCl 150mL EDTA 20mL

DTT 0.3mM

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2.3.2. SDS-Page

After 10 minutes incubation on 55°c to break secondary protein structures, 20µL of the samples are loaded on a 12% SDS-polyacrylamide in a chamber filled with 1x SDS-running buffer. For size determination 7µL of Precision Plus ProteinTM Dual Colour Standards (BioRad) was used. The power supply was set to 150V for 70 minutes.

12% running gel Stacking gel

Acrylamide 9,6mL Acrylamide 1,32mL Tris [1,5M pH 8.8] 6,0mL Tris [0,5M pH 6.8] 2,5mL SDS (10%) 240µL SDS (10%) 100µL APS [500mg/ml, 35%] 180µL APS [500mg/ml, 35%] 60µL ddH2O 7,72mL ddH2O 5,98mL TEMED 1,2µL TEMED 10µL

Table 7. SDS-PAGE gel components

10x SDS running buffer [1L] 1x SDS-running buffer [3L]

Tris 30g 10x SDS-running buffer 300mL

Glycine 154g ddH2O 2700mL

SDS 10g

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2.3.3. Western blot

After electrophoretic separation the proteins were transferred to an Immuno-Blot PVDF membrane (BioRad). The membrane was activation by immersion it in ethanol and a sandwich-Western blot assembly is constructed in the order (also see Figure 20):

Support-grid (towards cathode) Fiber-pad Filter paper PVDF membrane Gel Filter paper Fiber-pad

Support-grid (towards anode)

The Western-Blot assembly was placed into a chamber, surrounded with ice and filled with 1x blotting buffer. The power supply was set to 90V for 80 minutes.

Figure 20. Western-blot set-up: sandwich assembly is immersed in the blotting-buffer filled

chamber. [78]

10x Blotting buffer [1L; pH 8.3] 1x Blotting buffer [1L]

Tris 30,3g 10x blotting _buffer 100mL

Glycine 171g ddH2O 700mL

Isopropanol

[98,9% p.a.] 200mL

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2.3.4. Immunodetection

To block the unspecific binding sides of the PVDF-membrane a 5% milk powder PBS-solution is used. A rabbit anti-Orai1-antibody (Sigma; 1,0mg/mL) diluted 1:2000 in 5% milk-PBS solution was used as primary antibody, as secondary antibody an anti-rabbit antibody bound to a horse radish peroxidase (Sigma; 1,0mg/mL) diluted 1:5000 in PBS-T was used.

For this, the membrane was put on a shaker for 1 hour. After blocking, the following steps were carried out on a shaker:

1. Incubation with primary antibody (anti Orai1 produced in rabbit) for 1hour 2. Washing 3x 5 min with PBST-buffer

3. Incubation with secondary antibody (anti rabbit) for 1 hour. 4. Washing 3x 5 min with PBST-buffer

After washing, the blotting-membrane was incubated in 1ml of luminol (BioRad) and 1ml of peroxide (BioRad) for 2 minutes to catalyze the light reaction necessary for the development of the X-ray film (GE Healthcare). The X-ray film was fixated on the membrane, kept in a hypercassette and developed in the dark room, using a developer- (Ilford) and a fixating-solution (Ilford). 10x PBS [1L; pH 7.2] 1x PBS [1l] 1x PBS-T [1L] NaCl 80g 10x PBS 100mL 10x PBS 100mL KCl 2g ddH2O 900mL ddH2O 900mL Na2HPO4 20,1g Tween-20 1mL NaH2PO4 1,7g KH2PO4 2g

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2.4. Patch clamp measurements

Patch clamp electrophysiology experiments were carried out 24 hours after transfection of the HEK293 cells. Experiments were carried out with the support of Herwig Grabmayr.

Whole cell currents were measured with an Axiovert S100 TV inverted microscope (Carl Zeiss), an Axopatch 200B microelectrode amplifier (Axon Instruments), a WR-89 three dimensional aqua purificate micromanipulator (Narishige International), and a pE Fluorescence LED illumination system (CoolLED Limited).

To evaluate the data and to control of the microelectrode amplifier, WinWCP software package (v4.2.2, University of Strathclyde) was used.

The cells located on glass dishes were washed with extracellular solution without Ca2+_{to clean}

the cells from any remaining DMEM and a 10mM Ca2+_{solution was used to fill the cell bath}

chamber. Triethylene glycol diamine tetra acetic acid (EGTA) triggers passive Ca2+ store depletion during the measurement. Two Ag/AgCl electrodes serving as recording and reference electrode and a voltage ramp in the range of -90mV to 90mV for one second was applied every five seconds with a holding potential set to 0mV. The current amplitudes were recorded at -74mV, which is specific for Orai1 channels. For the correction of “normal” store-dependent currents, the current amplitude recorded shortly after the formation of whole-cell configuration has to be subtracted from all subsequent amplitudes. For constitutive currents, which show permanent Ca2+_{influx, 10mM Ca}2+_{solution with additional 10µM Lanthanum (La}3+_{) were added}

at the end of the experiment to inhibit the current.

For comparability of the experiments individual measurements where normalised by dividing all current amplitudes by the whole-cell capacitance.

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2.5. Confocal fluorescence microscopy

Confocal fluorescence microscopy experiments were carried out after 24-30 hours of HEK293 transfection at room temperature. All microscopy experiments were carried out with the support of Matthias Sallinger.

The cells were washed with 2mM Ca2+_{extracellular solution to remove excessive DMEM.}

The chamber was filled with extracellular solution. Depending on the type of experiment, various extracellular solutions containing 0mM Ca2+_{or 1µM Thapsigargin (TG), to induce store-depletion}

were used.

All buffers used for patch clamp as well as for confocal fluorescence microscopy experiments are listed in Table 11. The experiments were performed using two different setups: For localisation studies a CSU-X1 Real-Time Confocal System (Yokogawa Electric Corporation) fitted with two CoolSNAP HQ2 CCD cameras (Photometrics), a dual port adapter (dichroic: 505lp, cyan emission filter: 470/24, yellow emission filter: 535/30, Chroma Technology Corporation). An Axio Observer.Z1 inverted microscope (Carl Zeiss) with two diode lasers (445 and 515 nm, Visitron Systems) which was placed on a Vision IsoStation anti-vibration table (Newport Corporation). The VisiView software package (v2.1.4, Visitron Systems) was used for image generation. Due to cross-excitation and crosstalk during the measurements appropriate cross-talk calibration factors were determined for each construct. Threshold determination and background subtraction for correct imaging was done. Images and the corresponding data were calculated on a pixel to pixel basis with a custom-made software integrated into MATLAB (v7.11.0, The MathWorks, Inc.) which implemented the method published by Zal and Gascoigne with a microscope-specific constant G parameter of 2.75.

For NFAT studies (described in the Results part) a QLC100 Real-Time Confocal System (VisiTech Int.) was connected to two Photometrics CoolSNAPHQ monochrome cameras (Roper Scientific) and a dual-port adapter (dichroic, 505lp; cyan emission filter, 485/30; yellow emission filter, 535/50; Chroma Technology Corp.) was used to record fluorescence images. The system was connected to an Axiovert 200 M microscope (ZEISS, Germany) in conjunction with two diode lasers (445 nm, 515 nm) (Visitron Systems).75

For controlling and image acquisition a VisiView 2.1.1 software (Visitron Systems) was used. Cross-excitation and cross-talk events were corrected as described in the paragraph above for the localisation studies.

The images and corresponding data values were too calculated on a pixel to pixel basis with a custom-made software integrated into MATLAB (v7.11.0, The MathWorks, Inc.) which implementing the method published by Zal and Gascoigne utilising a different microscope- specific constant G parameter of 2.0 compared to the localisation data evaluation.

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Table 11. Components of the used solutions. Different experiments require various extracellular

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3. Results

Three cancer-related Orai1 mutations (A122T, N147S and P164H) from the cBioPortal database were structurally examined with western blotting (via a cysteine crosslinking assay), electrophysiological properties were measured with whole-cell patch clamp and effects on the transcription factor NFAT were observed with fluorescence microscopy studies.

After verification of the desired point-mutations, followed by transfection of the plasmids into HEK239 cells, the influence of the given mutations on the hOrai1 pore architecture as well as functional features were analysed.

Results from cysteine crosslinking experiments, patch-clamp electrophysiology and different confocal fluorescence microscopy measurements are shown in that order in the following chapters.

3.1. Cysteine-crosslinking results

Figure 21. Depiction of used marker (75 and 25 kDa in red) (left) and example western blot,

showing re-drawn marker (for visibility on the X-ray film. The Δ64 hOrai1 monomers have a mass of 23,7kDa and appear right below the 25kDa Marker

The blots were analysed with the software ImageJ (National Institute of Health). Rate of dimerization was calculated according to:

Equation 1: Calculation of dimerization-rate

Rate

_Dimers

=

Dimers

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Figure 22. Results of crosslinking experiments. The data was collected from three independent

experiments. Degree of dimerization was calculated using Equation 1. Significant differences between cancer related mutants and their corresponding single point mutants are indicated by

an asterisk.

Statistical evaluation was carried out using OriginLab Data Analysis Student’s t-test with a p-value of p< 0.05. Significant deviations could be observed for R91C + A122T, R91C + N147S, E106 + A122T and E106E + P164H. No significant deviations were found for all F99C mutants. All experiments were performed with HEK293 cells as model organism.

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3.2. Patch clamp results

To investigate the effects of the mutants related to functional electrophysiological characteristics on Orai1, whole cell patch clamp measurements were conducted. N-terminally labelled CFP-STIM1 proteins were coexpressed with N-terminally labelled YFP-Orai1 constructs. Current activation was measured over time with a gradual Ca2+ store depletion via EDTA. Inward currents were normalized to cell capacitance (pA/pF) for comparability between different cells.

Figure 23. Patch-clamp data of A122T (red), N147S (green) and P164H (blue) mutants as well

as wild type (black) data. The data was normalized to cell capacitance. Error-bars signify standard error of the mean (SEM), significant difference in the activation of the channel is

indicated by grey circles, regarding N147S and P164H.

No constitutive activation of the channels was identified with whole cell patch clamp. However, significant differences in the activation could be observed between the wildtype and N147S as well as wild type and P164H at 160s. The activation of the channel was slower, leading to decreased currents through the channel.

Statistical evaluation was carried out using OriginLab Data Analysis Student’s t-test with a p-value of p< 0.05. All experiments were performed with HEK293 cells as model organism.

0

50

100

150

200 -5

0 CFP-STIM1 + YFP-Orai1 mutants

I

(p

A

/p

F

)

Time (s)

wt

n = 10

A122T

n = 7

N147S

n = 6

P164H

n = 6

*

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3.3. Confocal fluorescence microscopy

3.3.1. Localisation Studies

HEK293 cells were transfected with CFP-STIM1-OASF fragment and the YFP-tagged hOrai1 mutations of interest to identify correct localisation of the proteins. The CFP-excitation was measured to detect membrane localisation as well as correct STIM1-Orai1 interaction. When OASF-Orai1 binding is unaffected, OASF translocates to the plasma membrane, indicating interaction with Orai1. Images were created using MATLAB (v7.11.0, The MathWorks, Inc.). Proper localisation for all Orai1 constructs could be observed.

Figure 24. Plasma membrane localization diagrams (left) and confocal fluorescence microscopy

images (right) for WT-Orai1 and the corresponding mutations of interest recorded in the cellular centres (nuclei visible) of depicted cells.

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3.3.2. Nuclear Factor of Activated T-Cells (NFAT) screen

Spinning disk confocal fluorescence microscopy was used to identify the nuclear translocation of the transcription factor nuclear factor of activated T-cells (NFAT). CFP-tagged NFAT constructs were cotransfected with Orai1 WT as well as the corresponding mutant constructs to identify the translocation of NFAT. The translocation was measured 24 hours after transfection. This approach identifies constitutively active Orai1 channels and constitutive Ca2+_{entry. The}

NFAT-signalling cascade is activated via STIM1-Orai1 mediated Ca2+ entry. Increased intracellular Ca2+ _{concentration} _{activates the Calmodulin/Calcineurin signalling} _cascade _that

dephosphorylates NFAT and leads to translocation of NFAT into the nucleus. This induces gene transcription. The percentage of cells with nuclear NFAT localization was determined using MATLAB (v7.11.0, The MathWorks, Inc.). [79]

Figure 25. Percentage of cells with nuclear NFAT localization. Measurements were conducted

24 hours after co-transfection with YFP-Orai1 constructs in 2mM Ca2+_{solution. No significant}

deviations were identified (t-test, p< 0.05).

WT A122T N147S P164H 0 5 10 15 20 25 30

cells with

nu

clear

NFAT

loca

lization

[%]

N=300 N=201 _N=211 N=196

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Figure 26. Representative NFAT fluorescence images. Images of cells coexpressing Orai1

mutant constructs (YFP labelled; left), CFP-NFAT (central) and overlay of the images (right). Nuclear NFAT localization is shown on the CFP pictures in green and Orai1 wildtype as well as

Characterization of cancer related Orai1 mutants A122T, N147S and P164H / submitted by Sascha Berlansky BSc