Proximity Ligation Assay for High Performance Protein Analysis in Medicine

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It is about wonder, discovery, and excitement.

To my family

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List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Liu Y., Gu J., Hagner-McWhirter Å., Sathiyanarayanan P., Gullberg M., Söderberg O., Johansson J., Hammond M., Ivansson D., Landegren U. Western blotting via prox- imity ligation for high performance protein analysis. Mol Cell Proteomics (2011), 10(11):01103-9.

II Gu J.G.*, Friedman M.*, Jost C., Johnsson K., Kamali- Moghaddam M., Plückthun A., Landegren U., Söderberg O. Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation. N Bio- technology (2012), [Epub ahead of print] *equal contribution

III Gu J.G., Wu D., Lund H., Sunnemark D., Kvist A., Mil- ner R., Exkersley D., Nilsson L., Agerman K., Landegren U., Kamali-Moghaddam. Elevated MARK2–dependent phosphorylation of Tau in Alzheimer’s disease analysed via proximity ligation. J Alzheimers Dis. (2012), in press.

IV Gu J.G., Lund H., Wu D., Blokzijl A., Clausson C., Von Euler G., Sunnemark D., Landegren U., Kamali- Moghaddam M. Roles of individual MARK isoforms in tau’s phosphorylation in Alzheimer’s disease revealed by proximity ligation. Manuscript.

Reprints were made with permission from the respective publishers.

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Related work by the author

Peer Reviewed Research Articles

Darmanis S., Nong R.Y., Hammond M., Gu J., Alderborn A., Vänelid J., Siegbahn A., Gustafsdottir S., Ericsson O., Landegren U., Kamali- Moghaddam M. Sensitive plasma protein analysis by microparticle- based proximity ligation assays. Mol Cell Proteomics, (2009), 9(2):

327-35.

Chen P.*, Gu J.*, A rapid measurement of rutin-degrading enzyme activity of tartary buckwheat seed. Food Bioprod Process, (2011), 89:81-5. * equal contribution

Teranishi Y., Hur J.Y., Gu J.G., Behbahani H., Kamali-Moghaddam M., Winblad B., Frykman S., Tjernberg,O.L. Erlin-2 is associated with active γ-secretase in brain and affects amyloid β-peptide production, Biochem Biophy Res Commun, (2012), 424(3): 476-81.

Friedman F., Gu J.G., Ren P., Hampe S.C., Törn C., Fex M., Landegren U., Lernmark Å., Kamali-Moghaddam M., Detection of GAD65 and GAD65-GADA immune complex in T1D and SPS pa- tients using proximity ligation assay, Manuscript.

Review Articles

Conze T., Shetye A., Tanaka Y., Gu J., Larsson C., Göransson J., Tavoosidana G., Söderberg O., Nilsson M., Landegren U. Analysis of Genes, Transcripts, and Proteins via DNA Ligation. Annu Rev Anal Chem, (2009), 2:215-39.

Nong R.Y., Gu J., Darmanis S., Kamali-Moghaddam M., Landegren U. DNA assisted protein-detection technologies, Expert Rev Prote- omics, (2012), 9(1): 21-32.

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Abbreviations

2DE Two-dimensional gel electrophoresis

AD Alzheimer's disease

Aβ β-amyloid peptide

BC O2-benzylcytosine derivatives

BD Binding domain

BG Benzylguanine derivatives BRET Bioluminescence resonance energy transfer CDK5 Cyclin-dependent kinase 5

cDNA Complementary DNA

CLIP domain A mutant of DNA repair protein O⁶-alkylguanine- DNA alkyltransferase

Co-IP Co-immunoprecipitation DARPins Designed ankyrin repeat proteins

DNA Deoxyribonucleic acid

DTT Ditiothreitol EGF Epidermal growth factor EIA Enzyme immunoassay

ELISA Enzyme-linked immunosorbent assay Fc region Fragment crystallization region

FRET Fluorescence resonance energy transfer GFP Green fluorescent protein

GSK-3 Glycogen synthase kinase 3 hCG Human chorionic gonadotropin

HER2 Human epidermal growth factor receptor 2 HRP Horseradish peroxidase IgA Immunoglobulin A

IgD Immunoglobulin D

IgE Immunoglobulin E

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IgG Immunoglobulin G

IgM Immunoglobulin M

kDa Kilo-Dalton LOD Limit of detection

MARK Microtubule affinity regulating kinase

MS Mass spectrometry

NFT Neurofibrillary tangle PCA Protein fragment complementation assay PCR Polymerase chain reaction

PDGF-BB Platelet-derived growth factor-BB PLA Proximity ligation assay

PVDF Polyvinylidene fluoride RCA Rolling circle amplification

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SNAP domain Another mutant of DNA repair protein O⁶- alkylguanine-DNA alkyltransferase

SPR Surface plasmon resonance

Sulfo-SMCC Sulfosucinimidul-4-(N-maleimidomethyl) cyclohexane-1-carbosylate

tRNA Transfer RNA

VEGF Vascular endothelial growth factor

Y2H Two-hybrid system

YFP Yellow fluorescent protein

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Introduction

The human genome contains about 20,000-25,000 protein-coding genes [1]

and encodes for more diverse and complex human proteome through for example splicing and post-translational modifications. The completion of the human genome project in 2003 greatly boosted human proteome but due to the great diversity and complexity in human proteome and protein interac- tomes, they require further investigation.

Molecular tools provide great opportunities for understanding the roles of proteins in health and diseases, such as in physiological and pathological signaling pathways, and development of advanced techniques has contribut- ed to improve protein analyses. The work presented in this thesis is a contribution to protein analyses based on proximity ligation with increased sensitivity and specificity and its application in medicine.

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Protein constitution

Post-translational modifications impart augmented or entirely novel protein functions that complicate proteome analyses [2]. Common post-translational modifications include phosphorylation, glycosylation, acetylation, and ubiq- uitination. These post-translational modifications affect protein activities, locations, interaction partners and their roles in different signaling cascades, and the patterns of these modifications may differ between states of health and/or sickness [3]. In biomedical technologies, post-translational protein modifications and the proteins themselves can influence assay performance, and they represent important analytical targets in research and diagnostics.

Antibodies

In eukaryotes, antibodies with a molecular weight of about 150 kDa recog- nize and bind foreign substances, such as intruding microorganisms, and trigger immune responses for cleaning up the intruders. Antibodies are produced by B cells, and each B cell generates its own unique variant of antibody [4]. The basic structural unit of an antibody molecule is composed of four polypeptide chains, two identical light (L) chains, containing approximately 220 amino acids, and two identical heavy (H) chains, usually containing around 440 amino acids: these four chains are connected by both non- covalent and covalent disulfide bonds to form a Y-shaped structure. Two antigen-binding surfaces in one antibody molecule are usually formed by co- operation between light and heavy chains. As there are two antigen-binding sites, bivalent antibody molecules can efficiently cross-link molecules with two or more antigen determinants, also called epitopes (Figure 1A). The flexible hinge regions in heavy chains enhance the antigen-binding efficiency of antibody molecules. In higher vertebrates, there are five classes of antibodies, IgG, IgA, IgD, IgM, and IgE. Subclasses of IgG and IgA also exist;

for example, IgG has four subclasses (IgG1, IgG2, IgG3, and IgG4). These classes and subclasses have distinct heavy chains with distinct conformations of the hinge and Fc regions that impart separate characteristics for each sub- class [5].

Biochemical or recombinant methods can create a number of derivatives, such as monovalent antibody fragments (Fab, 50 kDa) [6] and single chain fragments (scFv, 25kDa) [7], that are based on the conventional antibody but are smaller and have better stability.

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Affinity protein scaffold

In parallel with the production of intact antibodies and their derivatives, a variety of protein scaffolds for molecular recognition have been developed on the basis of the increasing availability of both structural and genomic data on proteins. A protein scaffold library can include billions of molecules and each library contains a constant scaffold and randomized residues, which differ from each other. With this tremendous variety, there is, in principle, the opportunity to select an affinity scaffold that specifically recognizes any given target [8] with higher stability than the antibodies and their derivatives [9]. Simultaneously, selection methods for selecting suitable affinity scaffolds, such as phage, ribosome, or RNA display [10], have been developed:

these methods can be used to select the scaffolds with similar affinity to those obtained by traditional hybridoma technology [11].

Figure 1. Schematics of a typical antibody molecule (A) and a typical DARPin (B).

Designed ankyrin repeat proteins (DARPins), a representative of the protein scaffolds, are derived from natural ankyrin repeat proteins [12]. The protein

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architecture of DARPins features different numbers of a structural motif stacked to form repeated protein domains, these are flanked by capping domains that shield the continuous hydrophobic core of the repeat domains [13-15] (Figure1B).

In many tumors, members of the epidermal growth factor (EGF) receptor family are highly overexpressed and form abnormal hetero- and homodi- mers, which alter the binding of ligands [16-18]. Although monoclonal antibodies, such as trastuzumab and pertuzumab, specific to HER2 protein are used for therapeutic applications in malignancy [19, 20], the use of antibodies as therapeutic agents has some limitations, such as inadequate pharmacokinetics, tissue penetration, and undesired interactions with the immune system [21]. Consequently, alternative affinity binders against EFG family members have been developed. In particular, DARPins selected for use against the EGF receptor family have impressive affinities in the pM to low nM range [22, 23], in particular, G3, a HER2-specific DARPin, has shown better correlation with HER2 amplification status in paraffin- embedded tissue sections than the FDA-approved antibody 4B5 [24].

Functionalization of affinity reagent for protein analysis

Nature offers opportunities for obtaining affinity reagents that can selectively catch protein targets and provides inspiration on how to transfer and report these selective recognitions into interpretable signals, such as linked enzymes, fluorescent groups, or the use of DNA as a reporter. For example, in the protein-primed DNA replication of adenovirus and bacteriophage, φ29 polymerase primes DNA replication through physically combining protein molecules with DNA strands [25].

Different techniques can couple affinity protein either with small molecules, such as fluorophores and isotopes, or with large polymers, such as oligonucleotides, and usually, bi-functional chemistry is used to conjugate oligonucleotides to proteins. For example, sulfosucinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carbosylate (Sulfo-SMCC) contains an amine-reactive N-hydroxysuccinimide (NHS ester) and sulfhydryl-reactive maleimide group. Sulfo-SMCC can readily couple thiol-modified oligonucleotides to antibodies through reactions with primary amines of antibodies, thereby functionalizing antibodies for protein analysis by DNA-assisted techniques [26, 27]. However, although this conjugation strategy is straight- forward, the antibodies undergo random coupling of oligonucleotides to any available primary amines, which sometimes adversely affects the affinity and binding ability of antibodies.

Ideally, affinity reagents should be functionalized at a single and specific site, so there is minimal influence on their affinity, and conjugated in a one- to-one ratio with report molecules. For example, after genetically modifying

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a single amino acid, anti-HER2 Fab is conjugated and functionalized at specific site and with homogeneity [28]. Small affinity binders, such as DARPins, are easy to genetically engineer for fusion with protein domains, and can be conjugated to report molecules via the protein domains. For instance, the SNAP domain, a mutant form of the human DNA repair protein O⁶-alkylguanine-DNA alkyltransferase, reacts rapidly and specifically with O⁶-benzylguanine (BG) and with derivatives that carry a large moiety (i.e.

oligonucleotide) linked to the benzyl group [29]. With guanine as a leaving group, the benzyl moiety becomes covalently attached to a cysteine residue at the specific site in the SNAP domain. The enzyme can be mutagenized to another form, the CLIP domain, which is specific for O⁶-benzylcytosine (BC) [30]. Both protein domains (about 20 kDa) and their vectors are com- mercially available from New England Biolabs [30-32].

Methods for protein analysis

Since 2000, the field of proteomics has witnessed major developments in the understanding of normal and pathogenic processes and the identification of potential drug targets for disease treatments. As a result, many methods for protein analysis have been developed and used in research laboratories and for diagnostics.

Two-hybrid system

The two-hybrid system (Y2H) was first developed in yeast cells by Fields et al in 1989 [33] and is one of the widely used methods for studying protein- protein interactions. The fundamental principle of Y2H is that signals from a downstream reporter gene are activated by a transcription factor bound to an upstream activation sequence. In a typical Y2H experiment, the transcription factor is divided into a DNA binding domain and an activating domain. Pro- tein X, referred to as the bait protein, is fused to the DNA binding domain, and protein Y, referred to as the prey protein, is fused to the activating domain. Interaction between the bait protein and the prey domain creates a physical proximity between the DNA binding domain and the activating domain, resulting in transcription of the reporter gene, such as transcription factor Gal4 [33]. Although technically Y2H can identify possible protein interactions, including direct and indirect interactions, there is a concern about the quality of the data and data annotation, such as numerous false positive annotations [34]. To detect post-modifications of protein such as phosphorylation, a mammalian variant Y2H has been established [35] and a cytoplasm-based Y2H variant has also been developed to study interactions occurring between integral membrane proteins [36-39]. In addition, Y2H

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variants, such as in bacteria, have been developed for screening protein interactions [40, 41].

Protein fragment complementation assay

The protein fragment complementation assay (PCA) was first implemented with a split ubiquitin. Ubiquitin is divided into two inactive fragments (Nub and Cub), each being fused to a bait protein or a prey protein. Depending on the interaction between the bait protein and the prey protein, the function of ubiquitin is reestablished through complementation of the two fragments, resulting in cleavage of the reporter protein by ubiquitin-dependent proteases [42, 43]. Other enzymes, such as galactosidase [44], dihydrofolate reductase [45], or lactamase [46], which converts chromogenic or fluoregenic sub- strates, are also used in this technique to screen protein interactions in vivo and in vitro. Although series of PCA upon fluorescent proteins are reported, for example, a split green fluorescent protein (GFP) [47] and a yellow fluorescent protein (YFP) [48, 49], PCA is prone to generate large numbers of artifacts due to indirect and delayed responses. For example, the folding and chromophore formation of fluorescent proteins is slow and maturation life- times of the complexes ranging between several minutes to hours are required [50].

Resonance energy transfer

With the establishment of the energy transfer theory [51] and auto- fluorescent proteins, the development of fluorescence resonance energy transfer (FRET) [52] was based on the radiation-less transfer of energy from a fluorophore in an excited state (donor) in close proximity (no more than 10 nm apart) to a second chromophore (acceptor). FRET readily senses a change in emission intensities of a donor and an acceptor, or a change in the life-time of fluorescence or photoluminescence [53].

To avoid cellular autofluorescence, photobleaching, and bias due to the direct excitement of the acceptors [54], bioluminescence resonance energy transfer (BRET) was developed. In BRET, bioluminescence plays the role of energy donor and green fluorescent protein (GFP) or/and yellow fluorescent protein (YFP) are used as acceptors, typically. As the readout is straightfor- ward and does not require external excitation, this technique provides a better perspective in high-throughput screening. However, the efficiency of both FRET and BRET are dependent on the proper orientations of both do- nor and acceptor dipoles and the distance between donor and acceptor [55].

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Enzyme-linked immunoassay

The enzyme-linked immunoassay is a valuable tool in medical laboratories for in vitro diagnostics and prognosis. The enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) was independently and simultaneously developed by Perlmann and Engvall (Sweden) [56, 57] and by Schuurs and Van Weemen (The Netherlands) [58]. In these assays, enzyme- labeled antibodies detect targets of interest (Figure 2). For instance, in indirect sandwich ELISA, antibodies specific to antigens, are immobilized on a solid support. After blocking, a sample containing antigens is incubated with the immobilized antibodies and the sample is then washed. Next, antibodies specific for the antigens are applied as primary antibodies, followed by washes. In the next step, enzyme-linked secondary antibodies are applied and these bind to specific primary antibodies, followed by washes. Sub- strates are then applied and converted by enzymes into colored or fluorescent products, and the resulting readout presents signals that correspond to the antigens in the sample. A classic example of ELISA is the pregnancy test [59]. In the pregnancy test, human chorionc gonadotropin (hCG) in urine samples can be detected with an accuracy of 86.9%, a diagnostic sensitivity of 90.6%, and a diagnostic specificity of approximately 100% [60].

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Figure 2. Schematics of different ELISA. (A) Direct ELISA. Enzyme-conjugated antibodies are added to antigens coupled to a surface. (B) Indirect ELISA. Antigens coated on a surface are recognized by primary antigen-specific antibodies. Enzyme- conjugated secondary antibodies are then added to target the primary antibodies. (C) Sandwich ELISA. Antigen-specific antibodies are coated on a surface to capture antigens. Enzyme-conjugated antibodies specific for the antigens bind to the captured antigens. (D) Indirect sandwich ELISA. The antibody-captured antigens are recognized by antigen-specific primary antibodies, which are then indirectly detected by enzyme-conjugated secondary antibodies specific for the primary antibodies added in solution. For all ELISA, the signals are produced via an enzymatic reaction that converts substrate molecules to colored, fluorescent, or luminescent catabolites.

Mass spectrometry

To meet the challenges of analyzing human proteomics (http://www.hupo.org), mass spectrometry (MS) rapidly became the method of choice for analyzing complex protein samples, such as post-translational modified proteins in tissue lysates [61-63].

MS takes advantage of tandem affinity purification, in which two distinct tags in fusion with a bait protein, the tag-fused protein, can be selectively purified from a complex biological matrix, together with any proteins associated with the bait protein. Subsequently, the purified and associated proteins are separated by gel electrophoresis and identified with MS [64-68].

However, affinity-tagged protein purification is biased to proteins interacting with bait proteins with high affinity and slow dissociation kinetics. Moreo-

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ver, after expression and during cell lysis, the bait protein can contact both physiological and non-physiological targets. As non-physiological targets might associate with bait protein or its protein complex [69], it is important eliminate contaminating proteins. Quantitative proteomics with metabolic or chemical labeling is a valuable tool for eliminating false positives [64]. For instance, two samples to be compared are labeled with light or heavy stable isotopes, then, the two samples are mixed, and the proteins from the combined samples are digested enzymatically. MS analyzes and distinguishes the heavy and light peptides according to their different masses. The ratio of ion abundance between the heavy and light peptides indicates the real abundance ratio of the peptide from the two samples, and the peptide can be identified via an open source program (MSQuant) [65].

Western blotting

Western blotting, first described by Burnette in 1981 [70], is an established protein analysis method for detecting protein targets. In western blotting, protein samples (i.e. tissue homogenates and cell culture lysates) are separated by gel electrophoresis, such as SDS-PAGE, native PAGE or two- dimensional gel electrophoresis (2DE) [71]. The separated proteins in the gel are transferred and immobilized to polyvinylidene fluoride (PVDF) or nitro- cellulose membranes, where the target proteins are visualized by specific probing with affinity reagents [72, 73]. After the membrane is blocked, the protein targets are detected by target-specific primary antibodies. This is followed by the addition of secondary antibodies labeled with reporting molecules, such as horseradish peroxidase (HRP) [74, 75], fluorophore [76], or quantum dots [77]. Semi-quantification of the protein targets is performed easily and digitally by densitometry or spectrophotometry [78, 79].

Array-based protein analysis

As an extension of DNA array, protein array is one of the landmarks in functional proteomics and interatome. Large numbers of well-defined capture molecules, such as antibodies, are immobilized on solid supports in a spatially defined manner, so called forward-phase protein microarray. Interacting partners that bind to the immobilized proteins are detected via fluorescence, radioactivity, or chemiluminescence. Microarray-based protein analyses enable the simultaneous study of thousands of protein targets [80]. However, compared to DNA analyses on microarrays, the scaling of protein analyses on microarray is more difficult; for example, one of the challenges in protein microarray is immobilizing large numbers of proteins with preserved physiological functions.

Surface plasmon resonance (SPR) detects soluble proteins through a reagent immobilized on the surface of a physicochemical transducer. One of the

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advantages of this method is that no labeling is required for detecting the proteins of interest. Moreover, real-time measurement is through equilibrium and interaction kinetics; therefore, binding kinetics with rate (1/s to 10^-4/s) and dissociation constants (100 pM to 100 µM) can be studied and quanti- fied [81]. Protein analyses on microparticles, such as the Luminex bead- based xMAP [82], are used for heterogeneous assays, and address the needs for multiplex assays and automation for high-throughput analysis [83] .

Proximity ligation assay (PLA)

Proximity ligation assay and its readout methods

DNA-assisted protein analysis has a relatively long history, with immuno- PCR [84] and immuno-RCA [85] as early examples. In these series of DNA- assisted protein analyses, the potential for high sensitivity has attracted attention from researchers. Powerful DNA amplification by polymerase chain reaction (PCR) [86, 87] (Figure 3A1 and A2) and rolling circle amplification (RCA) [88, 89] (Figure 3B1 and B2) can greatly improve detection sensitivity. The former technique provides exponential amplification and RCA offers linear and localized amplification, giving rise to a long, single-stranded DNA molecule. To improve detection specificity, combinations of several affinity reagents specific to protein targets are used in complex biological matrix e.g., human plasma proteomics where proteins concentrations differ by more than 10 orders of magnitude [90]. In the proximity ligation assay (PLA), two or more affinity binders are used to identify specific single protein, or its potential variants and modifications. Moreover, in PLA, powerful DNA amplification techniques, such as PCR and RCA, improve the limits of detection (LOD) [91, 92]. In the first PLA publication, DNA aptamers specifically directed against PDGF-BB were used as affinity reagents and gave rise to LOD as low as the attomol range on solid support: this detection sensitivity has also been demonstrated in complex biomaterial such as the detection of VEGF in whole blood samples [92]. Ligation- and DNA-assisted protein analyses have been thoroughly discussed from both historical and personalized medicine perspectives [90, 93].

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Figure 3. Two techniques for DNA-based readouts of protein analyses. (A1) In protein analyses, affinity reagents coupled with DNA first bind to protein molecules, the DNA on affinity reagents can then be amplified by PCR. (A2) The results of protein analysis can be recorded either by kinetic or by endpoint detection. (B1) Circular DNA strands that form due to protein detection reactions can be amplified by rolling circle amplification (RCA). (B2) RCA products can be observed for example as brightly fluorescent spots by hybridizing fluorophore-labeled oligonucleotides to the repeated sequence of the RCA products.

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Antibody–antigen interaction in PLA

The core of any immunoassay is the binding of analytes and antibodies.

Equilibrium of these binding events is desirable, but is not often achieved, particularly in rapid diagnosis of diseases and multiplex analysis of many different analytes. In these assays, it is important to understand the kinetics of antibody-analyte interactions. For example, PLA on solid support in- volves the capture for analytes by analyte-specific antibodies, followed by detection through at least two antibodies attached with DNA strands (Figure 4E).

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Figure 4. Protein analyses via proximity ligation. In situ PLA can detect expression levels of proteins (A), protein-protein interactions (B), and post-translational modifi- cations of proteins (C). In in situ PLA, protein targets are recognized by specific primary antibodies, followed by addition of secondary antibodies specific for the primary antibodies. The secondary antibodies are conjugated with different DNA strands, referred to as PLA probes. Upon simultaneous and proximal recognition of a protein target by two PLA probes, two other circularization DNA strands can hy- bridize to the DNA strands of the PLA probes. This allows the two DNA strands to be enzymatically ligated into a circular DNA template. Primed by an oligonucleotide on one of the PLA probes, the circular DNA can be amplified linearly with φ29

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polymerase and visualized by hybridization of fluorophore- or peroxidaselabeled detection oligonucleotides. In vitro, PLA can be used for protein analyses in solution (D) and on a solid support (E). Upon proximal binding by a pair of PLA probes to a protein target, the attached oligonucleotides can be ligated to form a DNA template, which can be recorded by quantitative PCR. As a variant of in vitro PLA, a proximi- ty extension assay (PEA) detects proteins and does not need DNA ligation (F) [94].

The reactions may take from seconds to a few hours to reach equilibrium, depending upon several parameters, such as temperature and pH value of the reaction. After being captured on microparticles or in fixed cells, the interactions between analytes and antibodies, to which the DNA strands have been conjugated, can be simplified through the law of mass action [95] as an equation below:

where: [Ag] is the concentration of analytes, [Ab] is the concentration of antibodies; [AgAb] is the concentration of complexes of antibody and antigen.

where: Ka is the association constant; Kd is the dissociation constant, and Keq is the equilibrium constant.

The equation 1.2 can be substituted…

…and rearranged:

where: [Abt] is the total concentration of antibodies ([Ab] + [AgAb]), and [Agb] is the concentration of bound antigens.

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This equation (1.5) implies a linear relation between [Agb]/[Ag] and [Agb], as reflected in a Scatchard plot [96]. In the equation (1.5), two parameters, Keq and [Abt], indicate the impact of equilibrium constants on the remaining analyte concentration (Figure 5A); alternatively, they represent the influence of [Abt] while maintaining the equilibrium constant (Figure 5B).

Figure 5. Scatchard plots of the effect of increasing the Keq while maintaining concentration of bound antibody (A) and the impact of changing antibody concentration (Abt) while maintaining the Keq constant (B).

Although PLA is typically used to detect analytes present in solution or immobilized on surfaces, several regarding analytes on surface and the change of reaction kinetics of interaction between analytes and PLA probes need to be considered. First, analytes immobilized on surfaces may lose critical epitopes because of conformational changes or from being predominantly hidden; new epitopes previously hidden may also arise. While antibodies immobilized on a solid surface or binding to analytes captured on a solid surface may also undergo conformational changes with respect to both vis-à- vis analyte-binding affinity and epitope-binding sites. As polyclonal antibodies are diverse, they are less influenced than monoclonal antibodies. Second, the reaction kinetics between analytes and antibodies is influenced by solid- phase immobilization of reagents [97]. The boundary layer of solid surfaces limits forward reaction of analytes and antibodies, therefore, the time to reach equilibrium changes [98]. In the boundary layer, reagents are not equally distributed on solid surfaces, such as analytes in fixed cells or immobilized via antibodies on microparticle beads, and clusters of analytes in the boundary layer can deplete antibody probes, such as PLA probes, rapidly and locally. Although this does not suggest the equilibrium constant per se increases it does imply low probability that dissociated probes can escape from the local high concentration of its potential interacting partner.

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Case study - tau phosphorylation in Alzheimer’s disease

Alzheimer’s disease (AD) was first described by Alois Alzheimer in 1907.

AD is the main cause of dementia and begins with impaired memory. The global prevalence of dementia is estimated to be as high as 24 million, and is predicted to double every 20 years until 2040 due to societal aging [99].

In order to understand the origin and pathological progression of AD at protein level and determine the underlying mechanism of AD, there has been intensive investigation of amyloid protein precursor, α-secretase, β-secretase, and γ-secretase. Amyloid precursor protein (APP) is abnormally cleaved by β- and γ-secretase to generate β-amyloid peptide (Aβ) that presents in two forms, Aβ₄₂ and Aβ₄₀. The toxicity of Aβ oligomers, especially Aβ₄₂,can eventually lead to apoptosis of neurons [71]. Mutant human APP molecules overexpressed in mice lead to Aβ deposition, this causes synaptic loss [100], synaptic dysfunction [101], memory loss [102] and inflammation [103].

Soluble Aβ oligomers may also affect phosphorylation of tau protein, thereby, influencing the generation of neurofibrillary tangles (NFT). Glycogen synthase kinase 3 beta (GSK-3 beta) and cyclin-dependent kinase 5 (cdk5) kinases have shown to regulate hyperphosphorylation of tau, and both kinases can be activated by soluble and extracellular Aβ [104-106]. However, in one study [107], where APP-overexpressing transgenic mice were crossed with tau-deficient mice, Aβ deposition was present in the offspring, but no dysfunction of memory was observed. The NFT in individual AD brains has demonstrated a better correlation with dementia than with amyloid deposition [108, 109].

Tau and tau phosphorylation in AD

In neurons, tau co-localizes with microtubules and maintains axonal integrity and regulates axonal transport [110]. In physiological processes, the phosphorylation of tau regulates tau’s affinity for binding to microtubules and further stabilizes the microtubules both spatially and temporally [111, 112].

In pathological processes of AD, tau is phosphorylated and then hyper- phosphorylated, thus, it becomes dislodged from the microtubules and ag- gregates into intraneuronal deposits known as NFT (Figure 6). Over 40 tau phosphorylation sites are serine-proline or threonine-proline motifs [113, 114], and mutational analyses of tau’s distinct phosphorylation sites identified three or four repeated KXGS motifs in the microtubule-binding domain of tau [115-118] which determine the binding affinity of tau to microtubules.

In vitro study [119] has revealed several kinases can phosphorylate KXGS motifs of tau, albeit most of them with low efficiency. Microtubule affinity regulating kinase (MARK), which was first purified from brain, efficiently detaches tau from microtubules and destabilizes microtubules in vitro and in

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cells. However, in AD it is unclear whether an increase of MARK activity or MARK expression takes place in response to the formation of intracellular NFT and loss of axonal-dentritic polarity [117].

MARK proteins belong to the calcium/calmodulin-dependent protein kinase group in the human kinome and consist of an N-terminal catalytic domain, an ubiquitin-associated domain, a spacer domain, and a tail domain.

The MARK family, consisting of four isoforms (MARK1-4), performs multiple functions, such as being responsible for embryonic polarity, and for the maintenance of polarity [117]. The current cascade hypothesis [117] for hyper-phosphorylation of tau in AD suggests MARK initiates a cascade for tau’s phosphorylation. MARK initially phosphorylates at KXGS motifs of tau and triggers dissociation of tau from microtubule surfaces, thus, the regions of tau containing the sites for proline-directed kinases become accessi- ble after dissociation of tau from microtubule, Then, CDK-5 and GSK-3 drive the hyper-phosphorylation of tau. The hyper-phosphorylated tau becomes smaller and prone to aggregation due to proteolytic cleavage and eventually form insoluble NFT [120]. In AD, eight or more phosphates per tau are present, whereas, in normal adult brain only one or two phosphates per tau are present [121]. However, whether proteolytic cleavage is required for aggregation is unknown, as the NFT does contain full-length tau [109].

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Figure 6. A proposed cascade in the formation of neurofibrillary tangles [120].

Inhibition of MARK: A potential therapy for Alzheimer disease?

Drug development for AD has mainly been driven by the amyloid hypothesis [122] and most drug candidates are directed to the Aβ₄₂ peptides, derived from APP by proteolytic cleavage of β- and γ-secretase. However, data from these drug candidates indicate the clearance of Aβ plaques is unlikely to reverse the damage or stop dementia in AD [123].

As NFT is one of the clinical hallmarks in AD, the development of drugs that interfere with relevant kinases for tau’s phosphorylation could potential- ly generate new therapeutics [124]. The inhibition of GSK-3 and Cdk5 [125], for instance by indirubin [126] and paullones [127] has received par-

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ticular attention. However, targeting of GSK-3 carries the risk of interfering with the Wnt signaling pathway [128], with possible harmful side effects.

Thus, as MARK plays an early role in phosphorylating tau, it may serve as an alternative target for preventing tauopathy in AD. For example, MARK4 can be considered as a drug target, as it has relatively higher brain expression than the other MARK isoforms [129]. However, with the sequence similarity of human MARK isoforms, the development of isoform-specific inhibitors may be problematic. Helicobacter pylori CagA, an inhibitor for associating microtubule protein (AMP), interacts with MARK isoforms and disrupts epithelial cell polarity [130], and derivative peptides from Helico- bacter pylori CagA that exhibit different affinities to MARK2 in vitro are selected [131].

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Present Investigations

The present investigations describe the developments and applications of the PLA as a highly selective and sensitive technique for analyzing proteins, protein-protein interactions and protein modifications on membrane in western blotting, in fixed cells, and on solid supports through either antibodies or alternative affinity reagent. Paper I reports improved protein analysis on western blotting through proximity ligation; paper II describes the use and performance of alternative affinity reagents in PLA; and papers III and IV present case studies as evidences for the medical applications of PLA.

Paper Ι: Western blotting via proximity ligation for high performance protein analysis

Aim of the study

As a globally used method for protein analysis, western blotting has limitations, such as detection specificity and sensitivity, and the detection of post- translational modifications of protein. The aim of the study in Paper I was to overcome these limitations and greatly improve the performance of protein analysis in western blotting via proximity ligation.

Experimental summary

Proteins present in lysates from cells or tissue, were separated by SDS- PAGE through gel pores towards an anode in an electrical field, and according to the migration rates of the SDS-coated protein. After SDS-PAGE, the separated proteins in the gel were electronically transferred and immobilized to PVDF membrane while maintaining their locations in the gel. The proteins were detected on membrane via proximity ligation.

After blocking the membrane, target proteins were detected with primary antibodies, as that in western blotting. For proximity ligation approach, spe- cies-specific antibodies covalently equipped with two different DNA strands, referred to as PLA probes, were added. Close colocalization of the targets allowed the two different DNA strands on two PLA probes to guide the formation of circular DNA templates through an enzymatic ligation reaction.

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Primed by the DNA strand of one of the PLA probes the circular DNA template was then locally amplified by phi29 polymerase to generate rolling circle amplification (RCA) products, which were concatemers of the com- plement of the circularized DNA strand. Individual RCA products were visualized through hybridization of oligonucleotides labeled with either fluorophore [89], or horseradish peroxidase (HRP), which generated the enzymatic production of colored precipitates [132, 133] or detectable via chemiluminescence.

Results and Discussion

This study published in 2011 has a technological focus, including extensive technical optimization and discussion on improving the performance of protein analysis, such as the choice of reaction chamber and conditions, back- ground reduction, readout methods, and its application for detecting protein modification without the need for stripping and re-probing as with traditional western blotting.

In this paper, the use of RCA enabled localized amplification of circular DNA formed in proximity ligation reactions, leading to a new achievement for detection sensitivity on blots. Taking advantage of the dual antibody recognitions, an increased specificity was demonstrated by only detecting the specific band using two cross-reactive anti-tubulin antibodies, each one of which produced distinct nonspecific bands in traditional western blotting.

As phosphorylated platelet-derived growth factor receptor β (PDGFβ) was detected with one primary antibody against receptor and the other primary antibody against phosphorylated tyrosine⁷⁵¹ residue, the need for stripping and re-probing the membrane or alignment of two separated blots, as in traditional western blotting, was avoided.

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Paper ΙΙ: Protein tag-mediated conjugation of

oligonucleotides to recombinant affinity binders for proximity ligation

Aim of the study

This study aimed to develop a convenient and robust approach to functional- ize recombinant affinity reagents in fusion with SNAP domains for DNA- mediated protein analysis, by attaching modified oligonucleotides to SNAP domains. With this approach, oligonucleotide could be conjugated at specific site on SNAP domain to yield precisely one oligonucleotide per affinity reagent. To study protein-protein interaction in situations where no high quality affinity binders were available, an in situ tag-mediated proximity ligation assay involving transfected fusion genes was developed.

Experimental summary

The SNAP domain is a genetically engineered mutant of the DNA repair O⁶ alkylguanine-DNA-alkyltransferase. Designed ankyrin repeat proteins (DARPins) were expressed in fusion with SNAP domains and the DARPin- SNAP was reacted in a one-to-one ratio with a BG-modified oligonucleotide at specific site to generate a PLA probe. Anti-HER2 DARPins were fused with SNAP domains and then conjugated to BG-modified oligonucleotides.

The resulting PLA probes were validated through PLA by visualizing the expression and interactions of endogenous HER2 proteins in human ovarian cancer cells and by measuring HER2 expression levels in lysates of breast cancer cells. The SNAP and CLIP domains were expressed in fusion with transfected genes, which allowed the conjugation of BG- and BC-modified DNA strands in situ for in situ PLA for visualizing the interactions between the expressed proteins, without the need for specific affinity reagents.

Results and Discussion

An efficient and robust method for attaching oligonucleotides to recombinant affinity binders via protein domains capable of covalently reacting with modified oligonucleotides was established. The DARPins were expressed in fusion with SNAP domains and these protein domains were used to connect BG-modified oligonucleotides to DARPins at specific sites and in one-to- one ratio to generate PLA probes.

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The PLA probes generated via SNAP domains in fusion with HER2-specific DARPins were used to detect HER2 expression and HER2 dimerization in fixed cells with in situ PLA, and to measure the expression level of HER2 in cell lysates by solid-phase PLA. Under optimal experimental conditions in situ and in vitro, the detection signals of HER2 was comparable and in agreement with the signals obtained with the monoclonal antibody pertuzumab in cells and commercial polyclonal antibodies in cell lysates. To demonstrate the possibility of conjugating SNAP- and CLIP- domain in situ with BG- and BC-modified oligonucleotides for in situ PLA, rapamycin- induced interactions between FKBP-SNAP and FRB-CLIP fusion proteins were analyzed and recorded, without requiring affinity reagents specific to FKBP and FRB.

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Papers ΙΙΙ & ΙV: Roles of all MARK isoforms in tau phosphorylation in Alzheimer’s disease revealed via proximity ligation

Aims of the studies

Both Papers III and IV aimed to analyze the roles of MARK isoforms in phosphorylating tau protein in AD. Paper III presents an analysis of MARK2-mediated phosphorylation of tau at the Ser²⁶² position and elevated interactions between MARK2 and tau in human AD brains investigated by proximity ligation. Paper IV describes a follow-up study of all MARK isoforms, and their roles in phosphorylating tau in AD, through proximity ligation.

Experimental summary

NIH 3T3 cells that stably expressed recombinant human 4R Tau (rhTau 3T3 cells) were transfected with individual MARK plasmids and a GFP- expressing plasmid. Isoform-specific antibodies, targeting for instance MARK2, were applied to each MARK-expressing rhTau 3T3 cells, followed by detection with PLA. With this approach, antibodies specific to MARK2 were selected. Through the similar approach, antibodies specific to individual MARK isoforms were selected.

The relation between individual MARK isoforms and tau in MARK- expressing cells was demonstrated through the use of staurosporine, a non- selective kinase inhibitor, and a synthetic CagA peptide, an inhibitor selective for associating microtubule protein (AMP), to inhibit MARK activity and the interaction with and phosphorylation of tau. In situ PLA in post- mortem human AD brains and non-demented elderly controls was performed to study the expression of individual MARK isoforms and their individual interactions with tau.

Results and Discussion

MARK2-specific antibodies were successfully identified by proximity ligation (Paper III). In the transfected cells, staurosporine significantly inhibited MARK2-tau interactions (p<0.001) and MARK2-mediated phosphorylation of tau at Ser²⁶² (p<0.001). In AD brains, PLA recorded significantly elevated

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interactions between MARK2 and tau (p<0.01) than that in non-demented elderly controls.

Isoform-specific antibodies against individual MARK isoforms were selected by PLA with cells expressing each of the MARK isoforms (Paper IV).

All MARK isoforms interacted with tau and caused phosphorylation of tau at Se²⁶². Staurosporine inhibited both the interactions and the phosphorylation of tau at Ser²⁶² by all MARK isoforms, however, the CagA peptide only inhibited tau’s phosphorylation by MARK4 (p < 0.0001), and not by other MARK isoforms. PLA was used to demonstrate MARK4-tau interactions were significantly elevated in the CA-field neurons of the hippocampus from AD cases than that in sections from elderly non-demented controls (p<0.0001 for the MARK4-tau interactions in CA2-CA1 field neurons, p<0.001 for the MARK4-tau interactions in CA4-CA3 field neurons). The elevated MARK4-tau interactions correlated with the Braak stage of AD. In conclusion, The MARK protein family, in particular MARK4, is a promising therapeutic target for AD, and PLA is a promising high-content screening method for drug entities.

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Acknowledgements

First of all, I would like to thank my opponent, Prof. Thomas Joos, and the committee board, Associate Prof. Amelie Eriksson Karlström, Associate Prof. Ann Brinkmalm, and Prof.Kjell-Olov Grönvik for reviewing my thesis and being in this dissertation.

I would like to extend my sincere gratitude to everybody who has supported me during my PhD studies in different ways. Especially, I would love to thank:

My supervisor, Ulf Landegren, whose expertise and optimism has created such an inspiring and creative research environment. Thank you for offering me this great opportunity of being here and for being supportive all the time.

Thank you for trusting me and encouraging me all the time. Millions thanks, dear Ulf.

My co-supervisor, Masood Kamali-Moghaddam, great thanks for believ- ing in me, your strong supports during these years, and for laughs, fun and interesting discussions.

My third ‘supervisor’, Ola Söderberg, thank you for your strong and posi- tive support and supervision on the interesting tag project! Thank you for being an impressive swimming coach and for teaching me how to swim! 

My examiner, Karin Forsberg Nilsson, thank you very much for your helpful advices and strong support, I always appreciate all the conversations we have had!

To former people in the molecular tools group, thank you all for all the great time over these years! Specially,

To Mikaela, thank you for your impressive organizing skills, being there with me for the tag project, for your patience on my illustration skills, and your encouragements et al. I appreciate very much for your listening and advices on both my scientific life and my personal life.

To members in morning-swimming team, Irene and Ida (I²), thank both of you for wonderful ballet time and introducing me to the nice riverside restaurant. Anna and Kalle, thank for the pleasant mushroom-picking time in the forest, and thank Lei and Tim for stopping me from picking and eating the beautiful and colourful mushrooms.

Sara, thank you for motivating me and accompanying me during our gym time. Chatarina, thank you for inspiring discussion during lunch time and introducing me to the exciting in situ padlock world; Yuki for your amazing sushi and the Japanese chocolate sticks; Jenny for being a wonderful of-

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ficemate and teaching me how to catch better images at the first place; Kate- rina for teaching me how to culture cells with great patience and your strong support for optimizing transfection efficiency; Lena L., special thanks for cheering me up through the cold and dark Swedish winters, thank you for your hugs in different occasions; Yanling Liu, thanks very much for great work in the western-PLA project and fun time together; Carolina for bringing me to the world of wonderful and amazing imaging analyses and all cell profiler seminars.

Thank, Mathias for showing me how to make a rotator and your super

‘candy’ blobs; Olle for introducing me to this group and giving me a guide tour with a detailed ‘projects’ map; Magnus and Henrik for sharing your great passion on the selector project and interesting discussions. Johan V for your positive attitude all the time, all office discussions and sharing!

Thank you for being supportive!

To present people in the molecular tools’ group, thank all of you for being here with me! Specially,

Christina and Liza, thank both of you for organizing my party (superb!!!

), nice and fun conversations during our fika. A special thank to Liza for proofreading my thesis and making me laugh in cell lab. Agata for the won- derful polish foods and sausages, fun discussion and chats about shoes and clothes, and science (of course  ); Karin for organizing after-work dinners and movies; Linda for gym time, you are an impressive and energetic train- ing coach! Caroline for valuable comments in group meeting, your seminar about patent issues, and being in the dimeric-probe project; Anja for the pleasant fika time in Linne café and our swimming time; Maria for keeping me accompany in Austria and Cambridge; Anna, my retreat roommate, thank you for interesting discussions during lunch, sharing me your great travelling experiences; Rachel for your inspiring presentations and com- ments in group meetings. Tonge thanks for being my nice neighbour in lab, I know it is not easy, particularly sometimes my used tips suddenly fly to your lab bench. Junhong, so enjoyable and lucky to have you sitting next to me in office and lab, hugs*hugs (you know what I mean  here)! Thanks for taking care of my apartment while I was away; Anne-Li, thank you for your hugs and the fun dancing time and introducing me to great dancing music;

Annika, you are an amazing friend! Thank you for being supportive and positive all the time! Lotta and Elin F for nice discussions, and your shar- ing on the two water taps in Britain (interestingly strong); Elin L for keeping our cell lab work and all your organizing efforts for this regarding; Elin E and Delal for keeping administrative things in order and for fun discus- sions.

Spyros, a special thank to you for making this group a more fun place with your acting skills! Carl-Magnus, thank you for running the chocolate bar that saves my afternoons and microscope time; Björn thank for all laughs and fun risen by your sense of humour; Andries, thank you for being

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supportive on my cell-culturing time, being positive when I was afraid of not being allowed to attend Switzerland (visa… a continuous issue); Malte for bring the Watsons’s water bottle back to lab and your comments about the brain images in my presentation; Rasel for spicing the lunch discussions up with your interesting opinions; David, thank you for being a great neighbour and for your numerous help and discussion on how to better present data, for tea time in Beijing airport and sushi in Tokyo, importantly, thank you for bringing me to the awesome paintball game; Di (Woody ),thank for being a great player in the paintball game (not my team member, ouch!) and for be- ing a motivating team player in lab (in the same team with me ); Lei for all interesting scientific and non-scientific discussions and wonderful time in and out of lab, and for driving me home in such changing summer weather here ; Johan O, thank you very much for saving my computer and helping me every time I panicked the sudden crash of my computer; Tomas, you are a calm and peaceful paintball player, I still do not understand how you put the black flag back, thank you for the fun time ! Erik for chairing this thesis defence, your introduction to many grants, organizing seminar and related events, thanks for your humoristic comments in lunch room . Mats, thank you very much for your support and tremendous contribution to this free and creative research environment, thank you for all the corridor meetings and discussions; Rongqin, thanks for the great party time and yummy hotpots!

for your passions on sharing and discussing ! Marco for your great and exciting sharing on the in situ sequencing and patience to my microscope questions! I also would like to thank Camilla, Monica and Joakim for con- tributing to this wonderful working environment.

Thank both of Ulla Steimer and Christina Andersson, for all your posi- tive energy, smiles, hugs, and support! Thanks very much for putting up with all kinds of questions from me.

-My students I have supervised and who have helped me in my projects over these years: VJ, thanks for your hard work on optimizing conjugation;

Aranzazu R. for your positive attitude and your hard work on γ-secretase project! 

-My collaborators, thank all of you very much for your great contribution to my projects! Special thanks to Harald Lund for your amazing and posi- tive attitude and support on MARK/tau project.

-To my friends out of lab, thank all of you for making my Uppsala life so fun! Specially, Larry Wu, thank you for making my Chinese better and con- fusing me with your name and Woody’s, I appreciate very much for your strong support! Yanling Cai, I always adore your cooking skills and loving your kitchen, Huahua,and Wangshu for girls discussions, foods and fun!

Nikki thank you for the fun course time and your positive attitude and sup- port! Xiang Jiao for keeping me accompany during swimming and for after- noon tea time; Sara, it was fun and cool to teach the lab section of the Fo- rensics course together with you! And Maria L for organizing the course and

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your smiles every day in the corridor! My French twin, Jen, it is great to find somebody born in the same day, and thank you for the fun nation time and the visiting time in Paris and all your sharing, hugs !

My family, Mamma and Pappa, thank you for believing in me all the time, for your support and love, for encouraging me to challenge my limits!

My superb Sister, you are the strongest woman I have ever known, thank you for your invaluable support and care through my life and deeply loving your little sister in all occasions.

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References:

1. Consortium IHGS: Finishing the euchromatic sequence of the human genome. Nature 2004, 431:931-945.

2. Jensen LJ, Gupta R, Blom N, Devos D, Tamames J, Kesmir C, Nielsen H, Staerfeldt HH, Rapacki K, Workman C, et al: Prediction of human protein function from post-translational modifications and localization features.

J Mol Biol 2002, 319:1257-1265.

3. Seo J, Lee KJ: Post-translational modifications and their biological functions: proteomic analysis and systematic approaches. J Biochem Mol Biol 2004, 37:35-44.

4. Ohara J, Paul WE: Production of a monoclonal antibody to and molecular characterization of B-cell stimulatory factor-1. Nature 1985, 315:333-336.

5. Horgan C, Brown K, Pincus SH: Studies on antigen binding by intact and hinge-deleted chimeric antibodies. J Immunol 1993, 150:5400-5407.

6. Rader C: Overview on concepts and applications of Fab antibody fragments. Curr Protoc Protein Sci 2009, Chapter 6:Unit 6 9.

7. Pansri P, Jaruseranee N, Rangnoi K, Kristensen P, Yamabhai M: A compact phage display human scFv library for selection of antibodies to a wide variety of antigens. BMC Biotechnol 2009, 9:6.

8. Bradbury AR, Marks JD: Antibodies from phage antibody libraries. J Immunol Methods 2004, 290:29-49.

9. Binz HK, Amstutz P, Kohl A, Stumpp MT, Briand C, Forrer P, Grutter MG, Pluckthun A: High-affinity binders selected from designed ankyrin repeat protein libraries. Nat Biotechnol 2004, 22:575-582.

10. Smith GP: Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985, 228:1315- 1317.

11. Kohler G, Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975, 256:495-497.

12. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, et al: Initial sequencing and analysis of the human genome. Nature 2001, 409:860-921.

13. Pancer Z, Amemiya CT, Ehrhardt GR, Ceitlin J, Gartland GL, Cooper MD:

Somatic diversification of variable lymphocyte receptors in the agnathan sea lamprey. Nature 2004, 430:174-180.

14. Kajander T, Cortajarena AL, Regan L: Consensus design as a tool for engineering repeat proteins. Methods Mol Biol 2006, 340:151-170.

15. Binz HK, Stumpp MT, Forrer P, Amstutz P, Pluckthun A: Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial

(41)

libraries of consensus ankyrin repeat proteins. J Mol Biol 2003, 332:489- 503.

16. Yarden Y, Sliwkowski MX: Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001, 2:127-137.

17. Yarden Y: Biology of HER2 and its importance in breast cancer.

Oncology 2001, 61 Suppl 2:1-13.

18. Yarden Y: The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities. Eur J Cancer 2001, 37 Suppl 4:S3-8.

19. Cobleigh MA, Vogel CL, Tripathy D, Robert NJ, Scholl S, Fehrenbacher L, Wolter JM, Paton V, Shak S, Lieberman G, Slamon DJ: Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol 1999, 17:2639-2648.

20. Adams GP, Weiner LM: Monoclonal antibody therapy of cancer. Nat Biotechnol 2005, 23:1147-1157.

21. Chames P, Van Regenmortel M, Weiss E, Baty D: Therapeutic antibodies:

successes, limitations and hopes for the future. Br J Pharmacol 2009, 157:220-233.

22. Pluckthun A, Binz HK, Amstutz P, Kohl A, Stumpp MT, Briand C, Forrer P, Grutter MG: High-affinity binders selected from designed ankyrin repeat protein libraries. Nature Biotechnology 2004, 22:575-582.

23. Pluckthun A, Zahnd C, Pecorari F, Straumann N, Wyler E: Selection and characterization of Her2 binding-designed ankyrin repeat proteins. J Biol Chem 2006, 281:35167-35175.

24. Theurillat JP, Dreier B, Nagy-Davidescu G, Seifert B, Behnke S, Zurrer- Hardi U, Ingold F, Pluckthun A, Moch H: Designed ankyrin repeat proteins: a novel tool for testing epidermal growth factor receptor 2 expression in breast cancer. Mod Pathol 2010, 23:1289-1297.

25. Salas M: Protein-priming of DNA replication. Annu Rev Biochem 1991, 60:39-71.

26. Hermanson GT: Bioconjugate techniques. San Diego: Academic Press; 1996.

27. Gustafsdottir SM, Nordengrahn A, Fredriksson S, Wallgren P, Rivera E, Schallmeiner E, Merza M, Landegren U: Detection of individual microbial pathogens by proximity ligation. Clin Chem 2006, 52:1152-1160.

28. Hutchins BM, Kazane SA, Staflin K, Forsyth JS, Felding-Habermann B, Smider VV, Schultz PG: Selective formation of covalent protein heterodimers with an unnatural amino acid. Chem Biol 2011, 18:299-303.

29. Gronemeyer T, Godin G, Johnsson K: Adding value to fusion proteins through covalent labelling. Curr Opin Biotechnol 2005, 16:453-458.

30. Gautier A, Juillerat A, Heinis C, Correa IR, Jr., Kindermann M, Beaufils F, Johnsson K: An engineered protein tag for multiprotein labeling in living cells. Chem Biol 2008, 15:128-136.

31. Maurel D, Comps-Agrar L, Brock C, Rives ML, Bourrier E, Ayoub MA, Bazin H, Tinel N, Durroux T, Prezeau L, et al: Cell-surface protein-protein

(42)

interaction analysis with time-resolved FRET and snap-tag technologies:

application to GPCR oligomerization. Nat Methods 2008, 5:561-567.

32. Kindermann M, George N, Johnsson N, Johnsson K: Covalent and selective immobilization of fusion proteins. J Am Chem Soc 2003, 125:7810-7811.

33. Fields S, Song O: A novel genetic system to detect protein-protein interactions. Nature 1989, 340:245-246.

34. von Mering C, Krause R, Snel B, Cornell M, Oliver SG, Fields S, Bork P:

Comparative assessment of large-scale data sets of protein-protein interactions. Nature 2002, 417:399-403.

35. Suter B, Kittanakom S, Stagljar I: Two-hybrid technologies in proteomics research. Curr Opin Biotechnol 2008, 19:316-323.

36. Luo Y, Batalao A, Zhou H, Zhu L: Mammalian two-hybrid system: a complementary approach to the yeast two-hybrid system. Biotechniques 1997, 22:350-352.

37. Fiebitz A, Nyarsik L, Haendler B, Hu YH, Wagner F, Thamm S, Lehrach H, Janitz M, Vanhecke D: High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays.

BMC Genomics 2008, 9:68.

38. Wehr MC, Laage R, Bolz U, Fischer TM, Grunewald S, Scheek S, Bach A, Nave KA, Rossner MJ: Monitoring regulated protein-protein interactions using split TEV. Nat Methods 2006, 3:985-993.

39. Eyckerman S, Verhee A, der Heyden JV, Lemmens I, Ostade XV, Vandekerckhove J, Tavernier J: Design and application of a cytokine- receptor-based interaction trap. Nat Cell Biol 2001, 3:1114-1119.

40. Joung JK, Ramm EI, Pabo CO: A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions. Proc Natl Acad Sci U S A 2000, 97:7382-7387.

41. Serebriiskii IG, Fang R, Latypova E, Hopkins R, Vinson C, Joung JK, Golemis EA: A combined yeast/bacteria two-hybrid system: development and evaluation. Mol Cell Proteomics 2005, 4:819-826.

42. Johnsson N, Varshavsky A: Split ubiquitin as a sensor of protein interactions in vivo. Proc Natl Acad Sci U S A 1994, 91:10340-10344.

43. Stagljar I, Korostensky C, Johnsson N, te Heesen S: A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci U S A 1998, 95:5187-5192.

44. Rossi F, Charlton CA, Blau HM: Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. Proc Natl Acad Sci U S A 1997, 94:8405-8410.

45. Remy I, Michnick SW: Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays. Proc Natl Acad Sci U S A 1999, 96:5394-5399.

46. Galarneau A, Primeau M, Trudeau LE, Michnick SW: Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions. Nat Biotechnol 2002, 20:619-622.

47. Cabantous S, Terwilliger TC, Waldo GS: Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein.

Nat Biotechnol 2005, 23:102-107.

(43)

48. Hu CD, Kerppola TK: Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol 2003, 21:539-545.

49. Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, Grill E:

Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science 2009, 324:1064-1068.

50. Stepanenko OV, Stepanenko OV, Shcherbakova DM, Kuznetsova IM, Turoverov KK, Verkhusha VV: Modern fluorescent proteins: from chromophore formation to novel intracellular applications. Biotechniques 2011, 51:313-+.

51. Stryer L, Haugland RP: Energy transfer: a spectroscopic ruler. Proc Natl Acad Sci U S A 1967, 58:719-726.

52. Haugland RP, Yguerabide J, Stryer L: Dependence of the kinetics of singlet-singlet energy transfer on spectral overlap. Proc Natl Acad Sci U S A 1969, 63:23-30.

53. Medintz IL, Clapp AR, Mattoussi H, Goldman ER, Fisher B, Mauro JM:

Self-assembled nanoscale biosensors based on quantum dot FRET donors. Nat Mater 2003, 2:630-638.

54. Pfleger KDG, Eidne KA: Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET).

Nature Methods 2006, 3:165-+.

55. Xu Y, Piston DW, Johnson CH: A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc Natl Acad Sci U S A 1999, 96:151-156.

56. Engvall E, Jonsson K, Perlmann P: Enzyme-linked immunosorbent assay.

II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes. Biochim Biophys Acta 1971, 251:427-434.

57. Engvall E, Perlmann P: Enzyme-linked immunosorbent assay (ELISA).

Quantitative assay of immunoglobulin G. Immunochemistry 1971, 8:871- 874.

58. Van Weemen BK, Schuurs AH: Immunoassay using antigen-enzyme conjugates. FEBS Lett 1971, 15:232-236.

59. Armstrong EG, Ehrlich PH, Birken S, Schlatterer JP, Siris E, Hembree WC, Canfield RE: Use of a highly sensitive and specific immunoradiometric assay for detection of human chorionic gonadotropin in urine of normal, nonpregnant, and pregnant individuals. J Clin Endocrinol Metab 1984, 59:867-874.

60. Christensen H, Thyssen HH, Schebye O, Berget A: Three highly sensitive

"bedside" serum and urine tests for pregnancy compared. Clin Chem 1990, 36:1686-1688.

61. Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM: Electrospray ionization for mass spectrometry of large biomolecules. Science 1989, 246:64-71.

62. Aebersold R, Goodlett DR: Mass spectrometry in proteomics. Chem Rev 2001, 101:269-295.

Proximity Ligation Assay for High Performance Protein Analysis in Medicine

It is about wonder, discovery, and excitement.

List of Papers

Related work by the author

Peer Reviewed Research Articles

Review Articles

Contents

Abbreviations

Introduction

Protein constitution

Antibodies

Affinity protein scaffold

Functionalization of affinity reagent for protein analysis

Methods for protein analysis

Two-hybrid system

Protein fragment complementation assay

Resonance energy transfer

Enzyme-linked immunoassay

Mass spectrometry

Western blotting

Array-based protein analysis

Proximity ligation assay (PLA)

Case study - tau phosphorylation in Alzheimer’s disease

Present Investigations

Paper Ι: Western blotting via proximity ligation for high performance protein analysis

Aim of the study

Experimental summary

Results and Discussion

Paper ΙΙ: Protein tag-mediated conjugation of

oligonucleotides to recombinant affinity binders for proximity ligation

Aim of the study

Experimental summary

Results and Discussion

Papers ΙΙΙ & ΙV: Roles of all MARK isoforms in tau phosphorylation in Alzheimer’s disease revealed via proximity ligation

Aims of the studies

Experimental summary

Results and Discussion

Acknowledgements

References: