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Biomolecular interaction analysis in functional proteomics

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Biomolecular interaction analysis in functional proteomics
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  DOI 10.1007/s00702-006-0515-5J Neural Transm (2006) Biomolecular interaction analysis in functional proteomics D. Moll 1 , A. Prinz 1 , F. Gesellchen 1 , S. Drewianka 2 , B. Zimmermann 2 , and  F. W. Herberg 1 1 Department of Biochemistry, University of Kassel, and 2 Biaffin GmbH & Co KG, Kassel, GermanyReceived September 25, 2005; accepted April 5, 2006Published online July 13, 2006; # Springer-Verlag 2006 Summary.  To understand the function of highly complex eukaryotic tissues like thehuman brain, in depth knowledge about cel-lular protein networks is required. Biomole-cular interaction analysis (BIA), as a part of functional proteomics, aims to quantify inter-action patterns within a protein network indetail. We used the cAMP dependent proteinkinase (PKA) as a model system for the bind-ing analysis between small natural ligands,cAMPandcAMPanalogues,withtheirphysio-logical interaction partner, the regulatory sub-unit of PKA. BIA comprises a variety of methods based on physics, biochemistry andmolecular biology. Here we compared side bysiderealtimeSPR(surfaceplasmonresonance,Biacore), a bead based assay (AlphaScreen),a fluorescence based method (Fluorescencepolarisation) and ITC (isothermal titrationcalorimetry). These  in vitro  methods werecomplemented by an in cell reporter assay,BRET 2 (bioluminescence resonance energytransfer), allowing to test the effects of cAMPanalogues in living cells. Keywords:  cAMP dependent protein kinase,surface plasmon resonance, fluorescencepolarization, isothermal titration calorimetry,bioluminescence resonance energy transfer,AlphaScreen. Abbreviations  AlphaScreen  amplified luminescence proxi-mity homogeneous assay;  BIA  biomolecularinteraction analysis;  BRET   bioluminescenceresonance energy transfer;  cAMP  adenosine-3 0 ,5 0 -cyclicmonophosphate;  C   catalytic sub-unit of PKA;  FP  fluorescence polarization;  ITC   isothermal titration calorimetry;  PKA cAMP dependent protein kinase;  PTM   posttranslational modifications;  R  regulatory sub-unit of PKA;  RU   response units;  SPR  surfaceplasmon resonance. Introduction The function of biological systems is me-diated by proteins and their interactions. Cel-lular activities are not only dependent onprotein expression patterns, but are also con-trolled by post translational modifications(PTMs), by compartmentalization and proteindegradation (Graves and Haystead, 2002). Inthe classical proteomics approach methodslike 2D gel electrophoresis and mass spec-trometry have been established to describeexpressionpatterns differentially e.g.compar-ing healthy and diseased tissue. One majorgoal of functional proteomics is to determineprotein–protein, protein-DNA, and protein-  ligand interaction with high accuracy and ingreat detail within a cellular network. Intra-cellular interaction pathways are influencedto a large extent by PTMs (Shaywitz et al.,2002; Yaqub et al., 2003). The most promi-nent PTM in an eukaryotic cell is proteinphosphorylation, a key event in the regula-tion of the cell mediated by the action of pro-tein kinases (Manning et al., 2002). About40% of all proteins may undergo this crucialPTM during some state of cellular growth anddifferentiation. The importance of proteinphosphorylation in signalling pathways is im-pressively reflected in the devastating effectsofproteinkinasedysfunctionlinkedto severalhuman diseases (Blume-Jensen and Hunter,2001; Sachsenmaier, 2001) most prominent-ly in human malignancies (Fabbro et al.,2002; Tasken and Aandahl, 2004). About518 Serine = Threonine and Tyrosine specificprotein kinases are encoded in the humangenome(Manningetal.,2002).Althoughpro-tein kinases differ substantially in their sub-strate specificity, activity, biological half life,cellular localisation and function, they sharea common overall protein fold. Cyclic AMPdependent protein kinase (PKA) has beenused as a model system for kinase functionduring thelast two decades and has been char-acterized biochemically and structurally indetail (Taylor, 1989; Knighton et al., 1991;Taylor et al., 2004). The enzyme consists of a regulatory (R) dimer and two monomericcatalytic (C) subunits forming an inactive ho-loenzyme complex (R 2 C 2 ). The holoenzymecomplex is activated by increasing concen-trations of the second messenger cAMP, alow molecular weight ligand. Upon bindingof cAMP to two distinct binding sites on eachR subunit, the C subunits are released (Fig. 1)and can phosphorylate substrates in the cyto-plasm, but they can also migrate into thenucleus, thus affecting gene regulation viathe cAMP response element binding protein(CREB (Hagiwara et al., 1993)). Not surpris-ingly, deregulation of PKA activity has beenimplicated in various human diseases, amongthem breast cancer, Carney complex andHIV infection (Tasken and Aandahl, 2004).PKA has received increasing attention as afactor contributing to neurological disorders.It has already been known that PKA activityis required for memory formation in themammalian hippocampus (Kandel, 2001).Recently, it was shown that PKA is alsoinvolved in processes concerning the workingmemory as well as reward-motivated learn-ing, linking PKA action potentially to mem-ory deficits and drug addiction (for review seeArnsten et al., 2005).Due to its modular structure the PKA sys-temisideallysuitedasamodelfortheanalysesof protein–protein interaction and protein-small ligand interaction. Here we analyzethe binding of the regulatory subunit of PKAto both the natural small ligand cAMP and tothe catalytic subunit.Several methods based on different phy-sico-chemical parameters are available to de-termine protein–protein interactions. Thesetechniques summarized under the term bio-molecular interaction analysis (BIA), allowan in depth look at molecular interactions.In the following we will describe interac-tions within the PKA model system deter-mined by surface plasmon resonance (SPR),AlphaScreen, fluorescence polarization (FP),isothermal titration calorimetry (ITC) andbioluminescence resonance energy transfer(BRET 2 ).Thesemethodscomprisesolidphase Fig. 1.  Model of PKA holoenzyme activation. Theinactive holoenzyme of cAMP dependent protein ki-nase (PKA) consists of a regulatory subunit dimer (Rsubunit, dark grey) and two catalytic subunits (C sub-unit, light grey). For simplification, only one C subunitinteracting with one R subunit monomer is shown.Binding of two molecules of cAMP (filled circle) toan R subunit monomer leads to the dissociation of theholoenzyme complex, thereby releasing the active CsubunitD. Moll et al.  assays as well as homogenous assays, per-formed  in vitro  and in living cells. Materials and methods  Reagents The synthetic peptide substrate Kemptide (LRRASLG)was purchased from Biosyntan GmbH (Berlin,Germany). ATP and NADH were obtained from Bio-mol GmbH (Hamburg, Germany).P11 cation exchanger cellulose and DE52 (Diethy-laminoethyl, DEAE) anion exchanger cellulose wereobtained from Whatman (Maidstone, UK).cAMP (adenosine-3 0 ,5 0 -cyclicmonophosphate),8AHA-cAMP (8-(6-aminohexyl)aminoadenosine-cAMP), 8CPT-cAMP (8-(4-chlorophenylthio)-cAMP),2Cl-cAMP (2-chloroadenosine-cAMP), 6AH-cAMP(N6-(6-aminohexyl)-cAMP), 8Fluo-cAMP  (8-[[2-[(fluoresceinylthioureido)amino]ethyl]thio]-cAMP),6Ph-cAMP (N6-phenyl-cAMP), 6MAH-cAMP (N6-(6-[N 0 -methylanthraniloyl]aminohexyl)-cAMP), 8Br-cAMP (8-bromo-cAMP), 8Cl-cAMP(8-chloro-cAMP),6MB-cAMP (N6-monobutyryl-cAMP), 8NBD-cAMP  (8-[[2-[(7-nitro-4-benzofurazanyl)amino]ethyl]thio]-cAMP), 8ADOA-cAMP (8-(8-amino-3,6-dioxaoctyla-mino)-cAMP), Sp8AHA-cAMP (8-(6-aminohexyl)aminoadenosine-3 0 ,5 0 -cyclicmonophosphorothioate, Sp-isomer), 8PIP-cAMP (8-piperidino-cAMP), 8AEA-cAMP (8-(2-aminoethyl)amino-cAMP), 2AHA-cAMP(2-(6-aminohexyl)amino-cAMP), 2 0 Mant-cAMP  (2 0 -O-(N-methylanthraniloyl)-cAMP), 8Br-cAMP-AM(8-Bromoadenosine-3 0 ,5 0 -cyclicmonophosphate, aceto-xymethylester), Sp8Br-cAMPS (8-bromoadenosine-3 0 ,5 0 -cyclicmonophosphorothioate), cGMP (guanosine-3 0 ,5 0 -cyclicmonophosphate) and cUMP (uridine-3 0 ,5 0 -cyclicmonophosphate) were obtained from Biolog LifeScience Institute (Bremen, Germany). Fine chemicals(research grade) were purchased from Roth (Karlsruhe,Germany) or from Sigma-Aldrich (Deisenhofen,Germany).CM5 sensor chips (research grade), NHS (N-hydro-xysuccinimide), EDC (N-ethyl-N 0 -(dimethylaminopro-pyl)-carbodiimide), ethanolamine-HCl, and surfactantP20wereobtainedfromBiacoreAB(Uppsala,Sweden). Preparation of recombinant proteins The cDNAs for the expression of recombinant pro-teins were kind gifts from Prof. S. S. Taylor, Universityof California, San Diego, USA (murine C a  and bovineR I a  1-91 (RI monomer)) and Prof. K. Tasken, Univer-sity of Oslo, Oslo, Norway (GST-hR I a  and hR II a ).RI monomer was overexpressed in  E. coli  BL21(DE3) and purified according to Herberg et al. usingion exchange chromatography (Herberg et al., 1994).GST-hR I a  was purified using Glutathion agarose fromSigma (Deisenhofen, Germany) following standardprotocols (Sambrook et al., 2001).To obtain cAMP free R subunits, the purified pro-tein was incubated with 10mM cGMP over night at4  C. Subsequently, excess cGMP was removed usinga PD 10 desalting column (Amersham Biosciences,Freiburg, Germany) followed by extensive dialysisagainst 150mM NaCl, 20mM MOPS, pH 7 (bufferA). To obtain completely nucleotide-free R subunitfor ITC measurements, the purified RI monomer wastreated with 8M urea to remove cAMP and then dia-lyzed as described above (Buechler et al., 1993).Recombinant PKA C subunit was expressed andpurified as described before (Slice and Taylor, 1989;Herberg et al., 1993).The purity of the R subunits was confirmed bySDS-polyacrylamide gel electrophoresis and the biolo-gical activity of the proteins was verified using thephosphotransferase assay with the peptide Kemptide asa substrate according to Cook et al. (1982).  AlphaScreen Biotin labelling of C subunit was performed with a 10:1molar excess of EZ-Link NHS Biotin (Perbio Sciences,Bonn, Germany) according to the manufacturer’s in-structions. The reaction was performed with intactholoenzyme, in order to protect the C subunit = R sub-unit interface from chemical modification during thebiotinylation procedure. After biotinylation, cAMP wasadded to the reaction mixture to dissociate the holo-enzyme complex and the free biotinylated C subunitwas subsequently purified using PKI(5–24) affinitychromatography (Olsen and Uhler, 1989).For the actual AlphaScreen measurements, biotiny-latedCsubunitandGST-taggedRsubunit(0.2nMeach)were mixed together in the presence of serial dilutionsof cAMP or Sp8Br-cAMPS in a 384 well microtiterplate (Optiplate, white, PerkinElmer). In a followingstep anti-GST acceptor beads and streptavidin donorbeads (PerkinElmer, Rodgau, Germany) were added toa final concentration of 20 m g = ml. The final reactionvolume in each well was 25 m l in 25mM Hepes, pH7.4, 100mM NaCl, 10mM MgCl 2 , 1mM ATP, 0.1%BSA. The resulting AlphaScreen signal (counts persecond) was determined with a Fusion TM a -FP micro-titerplatereader(PackardBioscience,nowPerkinElmer)after one hour incubation at room temperature. Fluorescence polarization (FP) Two different assay formats were used. In the directassay format both the RI monomer concentration aswell as the 8Fluo-cAMP concentration were variedBiomolecular interaction analysis  (10pM to 1 m M for RI monomer and 20nM to 500pMfor 8Fluo-cAMP). The assay was performed at 20  Cin buffer A containing 0.005% (v = v) CHAPS as asurfactant in a 384 well microtiterplate (Optiplate,black) using the Fusion TM a -FP microtiterplate reader.The fluorescence polarization signal was detected atEx 485nm = Em FP Filter 535nm with a PMT Voltageof 1100.For the fluorescence polarization displacementassay increasing concentrations of cAMP analogues(typically ranging from 1fM to 1 m M) were mixed with1nM 8Fluo-cAMP before adding the RI monomer.Here the concentration of the RI monomer was adaptedto 80% of the maximum value derived from the directassay using 1nM 8Fluo-cAMP. Fluorescence polariza-tion was measured after 5 minutes. Data were analyzedwith GraphPad Prism 4.0 (GraphPad Software, SanDiego, CA) by plotting the resulting polarization signalagainst the logarithm of the analogue concentration. Surface plasmon resonance (SPR) All SPR interaction analyses were performed at 20  Cin buffer A plus 0.005% (v = v) surfactant P20 using aBiacore 3000 instrument (Biacore AB, Sweden). Forcovalent coupling of 8AHA-cAMP, carboxymethylatedsensor chip surfaces (CM5, research grade) were acti-vated with NHS = EDC for 10 minutes and 8AHA-cAMP (3mM in 100mM HEPES, pH 8.0) was injectedfor 7 minutes with a flow rate of 5 m l = min. Deactivationof the surface was performed using 1M ethanolamine-HCl (pH 8.5) for 7 minutes. A reference cell wasactivated accordingly and deactivated subsequently.Competition analyses were performed by injection of 5nM RI monomer preincubated with varying concen-trations of each analogue to be analyzed. The bindingsignalwas monitoredfor 3minutes anddatapoints werecollected at the end of the association phase. The sensorsurfaces were regenerated after each binding cycle byseveral injections of 3M guanidinium HCl. After sub-tracting the reference cell, the resulting binding signalswere plotted against the logarithm of each cAMP ana-logue concentration and an EC 50  value was calculatedfrom the dose response curve using GraphPad Prism3.01 (GraphPad Software, Inc., San Diego = USA).  Isothermal titration calorimetry (ITC) The interaction between RI monomer and cGMP wasanalyzed in buffer A containing 1mM  b -mercaptoetha-nol using a VP-ITC microcalorimeter (MicroCal LLC.,Northampton, MA, USA). 5 m M RI monomer wereallowed to equilibrate in a 1.385ml cell at 20.0  C. Toensure that the titrant concentration was at its loadingvalue, two injections of51.3 m M cGMP (1 m l each) wereperformed before the actual titration experiment. 5 m linjections were conducted until no further binding wasobserved. Each time the reaction returned to baselinelevel (approximately after 3 minutes), a new injectionwas carried out. In order to minimize artifacts, cGMPwas dissolved in exactly the same buffer that was usedto dialyze the purified protein. For blank subtractioncGMP was injected in identical steps into buffer only.DataevaluationwasperformedwiththesoftwareMicro-Cal Origin for ITC (MicroCalLLC., Northampton,MA,USA; see also Wiseman et al., 1989), including correc-tions for volume change during the titration.  BRET  2 assay The human C a  coding sequence was amplified usingsense and antisense primers harboring unique  Hind  IIIand  BamH  I sites. The fragment was subcloned in-frameinto the  Hind  III =  BamH  I sites of pGFP 2 -C vector(PerkinElmer). The human R II a  coding sequence wasamplified without the stop codon allowing for cloningwith  BamH  I and  Kpn I. The fragments were subclonedusing the pTrcHis2-Topo + TA cloning kit (Invitrogen,Karlsruhe, Germany), excised and cloned in-frame into  BamH  I = Kpn I sites of pRluc-N vector (PerkinElmer).For BRET 2 assays, COS-7 cells were seeded ina white 96 well microtiterplate (Optiplate, White,PerkinElmer) at a density of 2  10 4 cells per well.Transfections were carried out in the microtiterplate24 hours later using 4 m l Polyfect reagent (Qiagen N.V.,Venlo, Netherlands) and a 0.5 m g plasmid DNA perwell and transfection. 48 hours post transfection, cellswere washed with glucose-supplemented Dulbecco’sPBS (D-PBS, Invitrogen), and the substrate Deep-BlueC(tm) (PerkinElmer) was added to a final concen-tration of 5 m M in a total volume of 50 m l D-PBS. Lightemission was detected using a Fusion TM a -FP mi-crotiterplate reader (PerkinElmer; read time 1 second,gain 50). The light output was taken consecutively foreach well using filters at 410nm wavelength (  80nmbandpass) for the donor and 515nm (  30nm band-pass) for the acceptor fluorophore. Background values,routinely obtained with untransfected cells, were sub-tracted in each measurement. Control transfections withempty pRluc and pGFP 2 vectors were carried outwith each experiment. BRET 2 signals were calculatedas (emission  515nm -background 515nm ) = (emission  410nm -background 410nm ). GraphPad Prism 4.0 was used forstatistical analysis. Results Biomolecular interaction analysis (BIA) isused to describe binding events between smallmolecules, proteins (e.g. receptors, enzymesand antibodies), peptides, nucleotides or car- D. Moll et al.  bohydrates. Here we focus on one hand on in-teraction between the PKA regulatory subunitand its low molecular weight ligand cAMPand on the other hand on the influence of cAMP on holoenzyme formation using four in vitro  methods and one in cell assay. Equi-librium binding data as well as associationand dissociation rate constants were obtained.  AlphaScreen allows compound screeningin a homogenous assay format  AlphaScreen is a bead-based proximity assay,where a luminescence signal is the readout fora biomolecular interaction. Both interactionpartners have to be chemically coupled tolatex beads (diameter 250nm). Upon illumi-nation with laser light ( l ex ¼ 680nm) singletoxygen  ð O 2 ð 1  g ÞÞ  is produced on the donorbeads (D), see Fig. 2 Inset. If a second inter-action partner, coupled to acceptor (A) beads,binds, both beads are brought into close prox-imity. The chemical energy of the singlet oxy-gen from the donor beads can diffuse to theacceptor beads, resulting in a luminescencesignal ( l em ¼ 520–620nm). If the interactionpartners do not bind to each other, the singletoxygen decays (t 1 = 2 ¼ 4 m s) and no lumines-cence signal is observed. The assay is usuallyperformed in a volume of 5–25 m l in 384 wellplates and is well suited for automation andthus for high throughput screening.Immobilization of the interaction partnersto donor and acceptor beads, respectively, canbe achieved by different strategies. Direct cou-pling of ligands containing primary aminescanbedoneviareductiveamination. However,in the case of PKA R and C subunits this Fig. 2.  Detection of PKA holoenzyme dissociation using AlphaScreen. Biotinylated C subunit and GST-tagged Rsubunit (0.2nM each) were incubated in a 384 well plate with increasing concentrations of cAMP and Sp8Br-cAMPS, respectively, before adding anti-GSTacceptor beads and streptavidin coated donor beads (20 m g = ml finalconcentration). Readings were taken after one hour incubation. Data were normalized and fitted according to asigmoidal dose-response model and EC 50  values were calculated. All data points represent the mean  SD of triplicate measurements. Inset: Depicted in the cartoon is the principle of an AlphaScreen assay. Donor (D) beadswere coupled with C subunits (light grey symbol), acceptor (A) beads with R subunits (dark grey symbol). Dbeads, containing a photosensitizer, release singlet oxygen  ð O 2 ð 1  g ÞÞ  upon illumination with laser light ( l ex 680nm). Due to the interaction of C subunits coupled to the donor beads and R subunits coupled to the acceptorbeads the chemical energy of the singlet oxygen is converted into a luminescence signal ( l em  520–620nm). If,upon addition of cAMP, the C and R subunits dissociate, the AlphaScreen signal diminishesBiomolecular interaction analysis
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