Proteomics Approach to Identify the Interacting Partners of Cellular

ARTICLE pubs.acs.org/jpr

Proteomics Approach to Identify the Interacting Partners of Cellular Prion Protein and Characterization of Rab7a Interaction in Neuronal Cells Saima Zafar,† Nicolas von Ahsen,† Michael Oellerich,† Inga Zerr,‡ Walter J. Schulz-Schaeffer,§ Victor W. Armstrong,†,|| and Abdul R. Asif *,† †

Department of Clinical Chemistry, ‡Department of Neurobiology, and §Department of Neuropathology, University Medical Center Goettingen (UMG), Robert-Koch-Strasse 40, 37075, Goettingen, Germany

bS Supporting Information ABSTRACT: The present study was undertaken to identify proteins interacting with PrPC that could provide new insights into its physiological functions and pathological role. Human PrPC was expressed in prion protein-deficient murine hippocampus (HpL3-4) neuronal cells. The PrPC along with its interacting proteins were affinity purified using STrEPTactin-chromatography, in-gel digested, and identified by Q-TOF MS/MS analysis. Forty-three proteins appeared to interact with PrPC in this neuronal cell line. Of these, 15 were already known for their interaction with PrPC or PrPSc, while 28 new proteins were identified. Interaction of a novel interacting partner of GTPase family-Rab7a, having a suggested role in vesicle trafficking, was further investigated using confocal laser scanning microscopy and reverse coimmunoprecipitation. Both reverse coimmunoprecipitation and immunofluorescence results confirmed potential interaction of Rab7a with the PrPC. siRNA against the Rab7a gene decreased expression of Rab7a protein, in PrPC expressing HpL3-4 and SH-SY5Y cells. This depleted Rab7a expression led to the enhanced accumulation of PrPC in Rab9 positive endosomal compartments and consequently an increased colocalization between PrPC/Rab9. However, the Rab9 accumulated PrPC remained sensitive to proteinase-K digestion. The work described demonstrated for the first time that Rab7a interacts with PrPC and highlighted the involvement of endosomal compartments in the trafficking and regulation of PrPC. KEYWORDS: prion protein, interacting protein, Rab7, siRNA

’ INTRODUCTION Cellular prion protein (PrPC) is a highly conserved protein throughout the evolution of mammals and therefore is thought to confer important but yet unidentified cellular functions.1 PrPC is expressed in most tissues, especially in the central nervous system and lymphoid tissues. Several functions has been hypothesized for PrPC including its ability to protect the cells against oxidative stress, influence the cell signaling mechanisms, and help keep the normal processing of sensory information by the olfactory system and overall neuronal survival.14 Bremer et al. has recently demonstrated that neuronal expression of PrPC is necessary for the maintenance of myelin sheath around peripheral nerves.5 Prions cause transmissible neurodegenerative diseases associated with accumulation of PrPSc, a misfolded and aggregated form of the PrPC. Prion-o-pathies are unique among neurodegenerative diseases because of their infectious potential.6 Protein misfolding is believed to be the cause of the prion disease, a process that can occur due to: (i) exposure to PrPSc or (ii) spontaneous misfolding initiated within the individuals due to yet not fully understood reasons.79 Infectious potential of misfolded prion proteins through interaction communicated to the native PrPC is strongly suggested to involve other r 2011 American Chemical Society

interacting proteins in the cell.10 Therefore, it is crucial to identify different partners with which PrPC might be associated in the cell. Endocytosis controls multiple cellular and physiological events and is getting widespread response as a signaling platform in addition to the plasma membrane. The Rab GTPase is a member of two of the RAS-related subfamilies that are believed to be involved in the regulation of endosome trafficking from formation, maturation, movement, and fusion/docking on target membranes.11 Similar to other RAS-related GTP-binding proteins, they cycle between GTP-bound active and GDP-bound inactive states.12 In humans, there are more than 60 members of the Rab family that are distributed to distinct intracellular compartments.13 Individual Rab proteins have been shown to act as hubs that regulate distinct trafficking steps temporally and spatially by facilitating the vesicle motility, tethering, and fusion.12 Rab7, a member of Rab family, is found associated with transport control from early to late endosomes, and from late endosomes to lysosomes.14,15 The active form of Rab7 facilitates lipid transport, degradation of nutrient transporter Received: March 2, 2011 Published: May 23, 2011 3123

dx.doi.org/10.1021/pr2001989 | J. Proteome Res. 2011, 10, 3123–3135

Journal of Proteome Research proteins, and the trafficking of ligandreceptor complexes involved in cell signaling.1618 PrPC cycles between the plasma membrane and endocytic compartments by utilizing both the clathrin- and nonclathrin-coated vesicles in the internalization of PrPC.19 Certain endosomal factors from small GTP binding RAS proteins (Rab, Ras, Rho, Sar1/Arf, and Ran) recruit PrPC in the intracellular trafficking pathways.11,13 The precise cellular functionality and trafficking of PrPC and what exact role small RAS-related GTP-binding proteins are playing in this trafficking are still not clear. In the present study, along with 42 other proteins, we were able to demonstrate that Rab7a interacts with PrPC using STrEPtag affinity based purification and mass spectrometry. The colocalization of PrPC with Rab7a using laser confocal microscopy and reverse coimmunoprecipitation further confirmed the interaction. Knocking down the Rab7a expression using siRNA led to the enhanced PrPC accumulation in the Rab9 positive endosomal compartments.

’ MATERIALS AND METHODS Mouse anti-PrP mAb 6H4 (Prionics AG, Zurich, Switzerland), SAF70 (SpiBio, Paris France), 3F4 (kind gift from Dr. Schmitz, UMG, Germany), Rabbit anti-Rab7 and Rabbit anti-Rab9 (Cell Signaling technology, Frankfurt, Germany), STrEP mAb-Classic (IBA, Goettingen, Germany), annexin A2 (BD Transduction, Heidelberg, Germany), annexin A5 (Abcam, Cambridge, U.K.), Vimentin (Dako, Hamburg, Germany), and actin cytoplasmic 1 (Sigma, Steinheim, Germany) were used as primary antibodies. HRP-conjugated rabbit antimouse pAb (IBA, Goettingen, Germany), antirabbit pAb (Santa Cruz Biotechnology, Santa Cruz, CA), goat antimouse cy3-conjugated (Dianova, Hamburg, Germany), goat antirabbit (Alexa 488-conjugated), and antimouse (Alexa 488-conjugated) were used as secondary antibody. Protease and phosphatase inhibitor cocktail (Roche, Mannheim, Germany) and Hoechst 33342 (Sigma-Aldrich,Steinheim, Germany). The mammalian expression vector pESG103 encoding the full length 1253 fused with One STrEP-tag at its C-terminus was generated with StarGate combinatorial cloning (IBA, Goettingen, Germany). Cell Culture, Gene Manipulation, Transfection and RNAi

HpL3-4 cells20 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich Chemie, Steinheim, Germany), supplemented with 10% fetal bovine serum (FBS) (Biochrom AG, Berlin, Germany), and 1% penicillin/streptomycin (PS) (Biochrom AG, Berlin, Germany) at 37 °C, 5% CO2 supply, and 95% humidity. SH-SY5Y (stably expressing PrPC) cells were cultured in DMEM, supplemented with 10% FBS, 1% PS, Geniticin 200 μg/mL, at 37 °C, supplied with 5% CO2, and 95% humidity. The cells were seeded in complete DMEM medium prior to transient transfection with plasmid and siRNA duplexes using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Rab7a siRNA duplex had a sense strand 50 -CUGCUGCGUUCUCCUAUUU-30 .21 Nontargeting siRNA duplex (control siRNA duplex negative control: Eurogentec) was used as a negative control. One STrEP-Tag Purification and Protein/Peptide Sequence Identification

Forty-eight hours after transient transfection, HpL3-4 cells were washed with cold 1 phosphate-buffered saline (PBS), scraped and centrifuged at 4 °C, ∼400 g for 20 min. The supernatant

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was decanted and the pellet was resuspended in cold 1 PBS and centrifuged again at 4 °C, ∼400 g for 10 min. Cells were lysed in lysis buffer (50 mM Tris-HCl, pH8, 1% Triton X-100, 0.5% CHAPS, 1 mM DTT, protease and phosphatase inhibitor cocktail). Cell lysates were homogenized with ultra sonication on ice and the lysates were centrifuged for 15 min with 543 000 g at 4 °C. Protein concentration was determined by the Bradford assay (Bio-Rad, Munchen, Germany). The STrEP-Tactin superflow beads were equilibrated by three times with washing buffer containing 100 mM Tris-HCl, pH8, 1% Triton X-100, 1 mM DTT, 150 mM NaCl, protease and phosphatase inhibitor cocktail. The total protein were diluted with wash buffer containing 100 mM Tris-HCl, pH8, 1% Triton X-100, 1 mM DTT, protease and phosphatase inhibitors up to final concentration of 0.1% CHAPS. Total protein (4 mg) was incubated with equilibrated STrEP-Tactin superflow beads for 1 h at 4 °C on a rocking platform and then centrifuge at 15 000 rpm for 2 min. Supernatant was removed and the STrEP-Tactin superflow beads with bound protein were washed 4 times with wash buffer and bound proteins were eluated with 2.5 mM desthiobiotin. The eluated proteins were precipitated with methanol/chloroform22 before separation on 1-DE SDS-PAGE. Immunoblot Analysis

Cell lysis and immunoblotting were performed as described previously. Briefly, cells were lysed (50 mM Tris-HCl, pH8, 1% Triton X-100, 0.5% CHAPS, 1 mM DTT), and lysates were cleared of cell debris (1 min, 1000 g, 4 °C). Cell lysates were supplemented with protease and phosphatase inhibitor (Roche) and were separated on 12.5% 1-DE SDS-PAGE. Expression of recombinant proteins was analyzed by immunoblot using antiPrP 6H4 monoclonal antibody (1:1000), anti-PrP SAF70 monoclonal antibody (1:5000) and anti-Rab7a mAb (1:1000), overnight at 4 °C. Membranes were then rinsed in 1 TBS-T and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (diluted 1:2000/1:5000) for 1 h at RT. Immunoreactivity was detected after immersion of the membranes into enhanced chemiluminescence (ECL) solution and exposition to ECL-Hyperfilm (Amersham Biosciences, Buckinghamshire, U.K.). Images were documented using the ScanMaker4 (Microtek, International), after correction for the background and band intensities were determined by densitometry using labImage (version 2.7.1, Kapelan GmbH, Germany) data analyzer software. Immunoprecipitation

Cell lysis was performed as described above and the insoluble cell debris was removed by centrifugation at 543 000 g for 15 min at 4 °C. Immunoprecipitation was performed using Dynabeads protein G, according to the manufacturer’s instructions. Samples of total cytoplasmic cell extracts or immunoprecipitated proteins (corresponding to 2 106 cells/lane) were subjected to 12.5% 1-DE SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore). Immunoblotting was performed as described above. In-Gel Tryptic Digestion and Peptide Sequence Analysis

Specific bands were excised from the silver-stained 1-DE gel into 12 mm2 slices, destained with 15 mM potassium ferricyanide/50 mM sodium thiosulfate (Aldrich/Sigma-Aldrich, Steinheim, Germany) and then equilibrated with 50 mM ammonium bicarbonate/50% acetonitrile (ACN) (Sigma-Aldrich) until destained. In-gel digestion was carried out according to a 3124

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Journal of Proteome Research

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Figure 1. Identification of PrPC multiprotein complex from HpL3-4 cells purification by C-terminus One STrEP-tag. TCL after 48 H of transient transfection was prepared and subjected to the C-terminus One STrEP-tag purification method. Aliquots from each step of the purification (starting material (TCL) 5%; flow-through (FT) 50%; washes 50%; eluates 25%) were analyzed by immuno blotting using (A) 6H4 and One STrEP-tag specific antibodies and D, M, and U correspond to the di-, mono-, and unglycosylated PrP. (B) Silverstained 1-DE, proteins identified by MS/MS analysis are listed in Table 1. (C) Confirmatory immuno blotting using 3F4, One STrEP-tag, and other interacting proteins specific antibodies.

previously described protocol.23 The extracted peptides were then dissolved in 0.1% formic acid (FA) for ESI-QTOF MS/MS. One microliter of tryptic digested peptide solution was introduced using a CapLC auto sampler (Waters) onto a μ-precolumn cartridge C18 pepMap (300 μm 5 mm; 5 μm partical size) and further separated through a C18 pepMap100 nano Series (75 μm 15 cm; 3 μm partical size) analytical column (LC Packings). The mobile phase consisted of solution A (0.1% FA in 5% ACN) and solution B (0.1% FA in 95% ACN). The single sample run time was set for 60 min. The chromatographically separated peptides were then analyzed on a Q-TOF Ultima Global (Micromass, Manchester, U.K.) mass spectrometer equipped with a nanoflow ESI Z-spray source in positive ion mode. The data acquisition was performed using MassLynx (v 4.0) software on a Windows NT PC and data were further processed on Protein-Lynx-Global-Server (v 2.1), (Micromass, Manchester, UK). Processed data were searched against MSDB and Swiss-Prot databases through the Mascot search engine using a peptide mass tolerance of (0.5 Da and fragment mass tolerance of (0.5 Da. The search criteria were set up to maximum one missed cleavage allowed by trypsin and protein modifications set to methionine oxidation and carbamidomethylcysteine, when appropriate. Immunofluorescence and Quantification Analysis

Cells were platted on chambered slides (Lab-Tek II; Thermo Fisher Scientific (Nunc GmbH & Co. KG), Langenselbold Site) and transfected with the C-terminus One STrEP-tag PrPC for 24, 36, and 48 h. They were subsequently washed in a 1 PBS and were fixed for 15 min with 100% ethanol. After fixation, cells were permeabilised with 0.2% Triton X-100 in 1 PBS, followed by 20 min blocking step using 0.2% casein-solution containing Tween 20. Co-localization of PrPC with interacting proteins was detected by applying the primary antibodies [anti-PrP 3F4 (1:200),

rabbit anti-Rab7a (1:50), and rabbit anti-Rab9 (1:50)] overnight at 4 °C. The monoclonal antibodies were detected by incubating slides for 60 min with Alexa 488 conjugated anti-rabbit (1:200), Alexa 488 conjugated anti-mouse (1:200) or Cy3-labeled antimouse secondary antibody (1:200). Incubation with Hoechst 33342 or with TO-PRO-3 iodide for 10 min was performed to visualize nuclei. Finally, coverslips were placed on the glass slides and mounted with Fluoromount (DAKO, Hamburg, Germany). All the steps were carried out in a dark humid chamber and were stopped by washing coverslips three times with 1 PBS and kept dry at 4 °C and in the dark. Control cells (not-transfected with C-terminus One STrEP-tag PrPC and transfected with empty vector) and RNAi-treated cells were harvested and processed under the same conditions as STrEP-tag PrPC transfected cells. Confocal laser scanning microscopy was carried out using LSM 510 laser-scanning microscope (Zeiss, G€ottingen, Germany; 488 nm Argon, 543 and 633 nm HeliumNeon excitation wavelengths) according to the manufacturer’s instructions for the localization of PrPC and other interacting proteins, using a 63/1.25 oil immersion lens. Individual images were analyzed separately for colocalization using LSM 5 (Zeiss) or ImageJ (WCIF plugin) software. For two-color analysis, stacks of images with a total thickness of approximately 30 μm were acquired, using a dynamic range of 12 bits per pixel. Co-localization expressed as a correlation coefficient indicates the strength and direction of the linear relationship between two fluorescence channels. Pearson’s linear correlation coefficient (rP) was used in this study to calculate fluorescence channel correlations. ELISA

ELISA (enzyme-linked immunosorbent assay) was performed using commercially available “BetaPrion” Bovine spongiform encephalopathy (BSE) EIA Test Kit according to supplier’s recommendations (Leipzig, Germany). 3125


P07356 Q8BFZ3

P18760

P26041

Q8VDD5

Q9WVA4

P68369

P99024 P20152

P62259

P63101

P14206

21 14

35

9

3

33

12

13 12

27

27

24

3126

P84078

P10107

P48036

Q60864

P51150

P08113

P17182

P05202

P05064

P16858

O09131

34

21

22

11

32

8

16

16

17

18

26

P63038

P60710

14

15

acc. no.

B.No

Glutathione S-transferase omega-1

GAPDH

aldolase A

Fructose-bisphosphate

mitochondrial

Aspartate aminotransferase,

Alpha-enolase

Endoplasmin

Ras-related protein Rab7a

phosphoprotein 1

Stress-induced-

Annexin A5

Annexin A1

ADP-ribosylation factor 1

protein

60 kDa heat shock

Laminin receptor 1

14-3-3 protein zeta/delta

14-3-3 protein epsilon

Tubulin beta-5 chain Vimentin

Tubulin alpha-1A

Transgelin-2

Myosin-9

Moesin

Cofilin-1

protein 2

Annexin A2 Beta-actin-like

Actin, cytoplasmic 1

protein name

Table 1. PrPC Interacting Partnersa

60.9

Known37

Novel

Novel

Novel

Novel

Novel

Novel

27.4

35.7

39.3

47.3

47.1

92.4

23.4

62.5

Novel

35.9

Known43

38.7

Novel

Novel

Known32

32.8

Known38,39

20.6

27.7

Known36

Novel

29.1

Known36

49.6 53.6

50.1

Known34

34

Known Novel

Known34

Known34

226.2

67.7

18.5

22.3

Known32

38.6 41.9

41.7

Known28

mass (kDa)

ligand

PrPSc

Novel

Novel

Novel

Novel

Known28 Novel

Novel

ligand

PrPC

Cytoskeleton: Cell growth/maintenance

Exhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activities

Role in glycolysis

Role in glycolysis

Role in amino acid metabolism, facilitates cellular uptake of long-chain free fatty acids

activator of plasminogen on the cell surface of leukocytes and neurons

Multifunctional enzyme, role in glycolysis, growth control, hypoxia tolerance, allergic responses, serve as a receptor and

Metabolism: Energy pathways

associated degradation (ERAD), ATPase activity

Molecular chaperone, processing and transport of secreted proteins, endoplasmic reticulum

Involved in late endocytic transport

Mediates the association of the molecular chaperones HSC70 and HSP90

Anticoagulant protein, indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade

Calcium/phospholipid-binding protein, promotes membrane fusion and is involved in exocytosis This protein regulates phospholipase A2 activity

Involved in protein trafficking among different compartments

conditions in the mitochondrial matrix

Facilitate the correct folding of imported proteins, prevent misfolding and promote the refolding under stress

Receptor for the cellular prion protein and the pathogenic prion protein

Adapter protein in signaling pathway

Adapter protein in signaling pathway

Cell communication: Signal transduction

Major constituent of microtubules Class-III intermediate filaments, found in various nonepithelial cells

Major constituent of microtubules

Muscle organ development

Role in cytokinesis, cell shape, secretion and capping

Probably involved in connections of major cytoskeletal structures to the plasma membrane

Controls reversibly actin polymerization, depolymerization and major component of intranuclear and cytoplasmic actin rods

Calcium-regulated membrane-binding protein Role in cell motility

Highly conserved, involved in cell motility

function (ExPASy)

Journal of Proteome Research ARTICLE


P20029

Q61753

P07901

P11499

P06151

13

9

9

20

Q9EQU5

18

10

P38647

10

P10126

P19324

16

14

P27773

12

P17742

P09103

12

36

P58252 P63017

6 10

Q99LP6

P35700

33

33

acc. no.

B.No

protein name

3127

dehydrogenase

A chain

L-lactate

90-beta

Heat shock protein HSP

Heat shock protein HSP 90-alpha

dehydrogenase

D-3-phosphoglycerate

regulated protein (Bip)

78 kDa glucose-

1-alpha 1

Elongation factor

cistrans isomerase A

Peptidyl-prolyl

1, mitochondrial

GrpE protein homologue

Protein SET

Known55

Novel

Novel

Novel

Known54

Novel

Novel

xNovel

Novel

Novel

mitochondrial

Novel

shock protein) Stress-70 protein,

Novel

Novel

Novel Novel

Novel

ligand

PrPC

Serpin H1 (47 kDa heat

isomerase A3

Protein disulfide-

isomerase

Protein disulfide-

71 kDa protein

Elongation factor 2 Heat shock cognate

Peroxiredoxin-1

Table 1. Continued

Known54

Known54

Novel

ligand

PrPSc mass

36.4

83.2

84.7

56.5

72.3

50

17.9

24.4

33.3

73.4

46.5

56.6

57.1

95.2 70.8

22.1

(kDa)

function (ExPASy)

Role in glycolysis

Molecular chaperone with ATPase activity

Molecular chaperone with ATPase activity

Amino-acid biosynthesis, serine biosynthesis

Oxidoreductase, Stress response

Role in facilitating the assembly of multimeric protein complexes inside the ER

Lipopolysaccharide binding; ATP binding

Promotes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis

Cell cycle

Accelerate the folding of proteins, catalyzes the cistrans isomerization of proline imidic peptide bonds in oligopeptides

Essential component of the PAM complex, control the nucleotide-dependent binding of mitochondrial HSP70 to substrate proteins

Protein folding

Involved in apoptosis, transcription, nucleosome assembly and histone binding

Regulation of nucleic acid metabolism

Implicated in the control of cell proliferation and cellular aging, also act as a chaperone

Binds specifically to collagen, involved as a chaperone in the biosynthetic pathway of collagen

Catalyzes the rearrangement of S-S- bonds in proteins

Catalyzes the formation, breakage and rearrangement of disulfide bonds

Role in GTP-dependent translocation of the nascent protein chain from the A-site to the P-site of the ribosome Chaperone

Protein metabolism

of growth factors and tumor necrosis factor-alpha, and regulates GDPD5 function

Involved in redox regulation, eliminating peroxides generated during metabolism, participate in the signaling cascades

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Role in glycolysis, fatty acid biosynthesis, gluconeogenesis, lipid synthesis 26.6 Novel mitochondrial

Triosephosphate isomerase P17751 31

Interacting proteins identified by C-terminus One STrEP-tag purification were identified by Q-TOF MS/MS analysis. The biological functions are assigned as in ExPASy protein database and Human Protein Reference Database.24 B.No = band number listed in Figure 1B; acc. no. = Swiss-Prot accession number; PrPC ligand = previously identified as a PrPC interacting partner; PrPSc ligand = previously identified as a PrPSc interacting partner. The detailed list of interacting proteins with score, peptide match, sequence coverage and sequences can be found in Supplementary Table 1, Supporting Information.

Role in glycolysis, oxidation reduction 35.5 Novel P08249 20

Malate dehydrogenase,

mass

(kDa) ligand acc. no. B.No

protein name

ligand

PrPSc PrPC

Table 1. Continued

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a

function (ExPASy)

Journal of Proteome Research Proteinase-K (PK) Degradation Assay

The total cell lysates (TCLs) (siRNA treated and nontreated) were incubated for 60 min with shaking at 37 °C in the presence of PK (50 μg/mL). The digestion was stopped by adding electrophoresis sample buffer and the PK resistant PrP was examined by Western blotting. Statistical Analysis

All results in this study were obtained from at least four independent sets of experiments and were expressed as mean ( SD using descriptive statistics. Densitometric analysis of 1-DE gels were performed using Labimage (Kapelan, Leipzig, Germany) correlation software.

’ RESULTS Interacting Partners of PrPC

Mammalian expression vector encoding C-terminus One STrEP-tag PrPC was established with combinatorial cloning in order to purify the interacting partners of PrPC. The HpL3-4 cells lacking endogenous PrPC were transiently transfected with the vector containing One STrEP-tag PrPC (PrPþ/þ) or control vector without PrPC construct (PrP/). Immunoblotting with PrPC and One STrEP-tag antibody confirmed its expression in transfected cells (Supplement Figure 1, Supporting Information). The PrPC localization was further confirmed by using anti-One STrEP-tag antibody which showed a complete overlapping with PrPC localization (Supplement Figure 2, Supporting Information). The PrPC was observed to be evenly distributed in the cytoplasm and on membrane, with highest membrane expression observed 48 h after transfection. STrEP-Tactin superflow beads were used to purify PrPC along with its interacting proteins from the TCLs (PrPC containing or empty vector transfected). Eluates were separated on 1-DE, electrotransferred to a PVDF membrane, and detected with 6H4 PrPC antibody as well as One STrEP-tag antibodies (Figure 1A). Following positive confirmation, the remaining eluate was 1-DE separated and silver nitrate stained (Figure 1B). Whole lane from PrPþ/þ and PrP/ transfected eluate were excised and in-gel digested, and proteins were identified by Q-TOF MS/MS analysis. All of the proteins identified from the PrP/ lane bands were considered as background contaminants and subtracted from the list of proteins identified from PrPþ/þ transfected eluate. Both known and novel PrPC interacting partners were among the identified proteins in this study (Table 1). Nine out of 43 proteins are already described as interacting partner of PrPC by pervious studies (Table 1). Two of them (tubulin alpha-1A and tubulin beta-5 chain) are also known for their interaction for PrPSc. However, 5 other proteins (actin, cofilin-1, GAPDH, D-3-phosphoglycerate dehydrogenase and heat shock protein 90-alpha) from our identified proteins are described as interacting partners for PrPSc but not for PrPC. Collectively, 23% of the interacting partners of PrPC identified in this study are related to cytoskeleton-cell growth/maintenance, 23% to cell communication and signal transduction, 14% to metabolism/energy pathways, 14% to protein metabolism, 14% to oxioreductase/stress response, and 5% to protein folding proteins. The remaining 7% proteins fall into three functional groups: “cell cycle, ATP binding and nucleic acid metabolism proteins”24 (Figure 2). A selection of identified proteins was further validated by immunoblotting using the proteinspecific antibodies from purified One STrEP-tag eluate. PrPC and STrEP-tag signal was detected in TCL and One STrEP-tag eluate from PrPþ/þ or PrPC. Rab7a, annexin A2, annexin A5, actin 3128



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Figure 2. Functional categorization of identified interacting partners of PrPC. Interacting proteins identified by C-terminus One STrEP-tag purification were identified by Q-TOF MS/MS analysis. The biological functions are assigned as in ExPASy protein database (http://expasy.org/) and Human Protein Reference Database.24

confirmed that Rab7a was coprecipitating the PrPC, providing additional evidence for the interaction. Rab7a and PrPC

Figure 3. PrPC interacts with Rab7a. (A) TCL (Lane 1 positive control). TCL were coimmunoprecipitated (IP) with 3F4 PrPC (Lane 2 PrP/ transfected and lane 3 PrPþ/þ transfected), Rab7a (lane 4 PrP/ transfected and lane 5 PrPþ/þ transfected), and immunoblotted with SAF70 PrPC antibody. (B) IP with 3F4 PrPC (Lane 1), Rab7a (lane 2) and were immunoblotted with Rab7a antibody.

cytoplasmic 1, and vimentin were only detected in PrPC One STrEPtag eluate. No signal was detected in the control purified eluates (Figure 1C). First evidence of PrPC interaction with Rab7a was originated from Q-TOF MS/MS identification of PrPC copurified eluates. The observations were subsequently confirmed by immunoblotting the One STrEP-tag purified eluate individually with Rab7a antibody (Figure 1C). No signal was detected in control purified eluates using PrPC antibody from total TCL of empty vector transfected cells. In order to further confirm the observation from One STrEP-tag purification system, TCL prepared from transiently PrPC transfected neuronal HpL3-4 cells were separately immunoprecipitated with Rab7a antibody using G-protein coupled magnetic beads (Figure 3). Eluate from this reverse coimmunoprecipitation was immunoblotted with SAF70 PrPC antibody to reconfirm the PrPC interaction (Figure 3A). Figure 3A, B shows the immunoprecipitation results with PrPC and Rab7a antibodies and subsequent confirmation by immunoblotting. Positive PrPC signal detected from 27 to 37 kDa

One STrEP-tag purification and coimmunoprecipitation assays provided evidence that Rab7a might be an interacting partner of PrPC. To further check the potential interaction and influence of Rab7a on PrPC localization and expression, PrPC was transiently expressed in HpL3-4 PrPC knockout cells. In addition, SH-SY5Y cells stably expressing PrPC were examined. PrPC showed colocalization with Rab7a in the cytosolic area. To quantify the extent of colocalization, Imagej (WCIF plugin) software was used. Colocalization in fluorescence imaging characterizes the overlap extent between two different fluorescent labels with different emission wavelengths. The detection of fluorescence signals from two differently labeled proteins within the same voxel (three-dimensional pixel) determines that these proteins are located in the same area or very near to each other. Two perfectly colocalized fluorescence signals, each displayed on separate x and y axes, will generate a scatter plot wherein the points fall in a line at 45° to either axis. In the situation of non-colocalized molecules, the resulting scatter plot reveals each color along its own axis, with no overlap at 45°. Quantification of colocalization of Rab7a and PrPC, using the distribution of fluorescence intensities in the scatter plots, showed a partial colocalization between Rab7a and PrPC (Figure 4). Pearson’s correlation coefficient rp (1e rp e1) was used to measure the relatedness of two fluorescence channels, where values of 0 indicate no relatedness, whereas values >0 indicate a relatedness between the two fluorescence channels. On the basis of positive correlation coefficients for all analyzed pairs of fluorescence channels, further calculations were permissible for colocalization coefficients, M1 and M2, which express the contribution of each fluorescence channel to the pixels of interest. Values of colocalization coefficients range between 0 and 1. A value of 0 indicates that none of the signal within thresholds in that channel colocalizes with the other channel. A value of 1 indicates that the entire signal within thresholds in that channel colocalizes with the 3129



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Figure 4. Effect of Rab7a depletion on PrPC localization. HpL3-4 PrPC knockout cells were treated with siRNA duplex (100 nM) to target Rab7a and PrPC. Forty-eight hours after transfection, PrPC and Rab7a expression was analyzed by using 3F4 anti-PrPC (red) and anti-Rab7a (green) antibodies. HpL3-4 PrPC knockout cells transfected with (A) PrP/ and nontargeting siRNA (negative control), (B) PrP/ and Rab7a siRNA, (C) PrPþ/þ and nontargeting siRNA, and (D) PrPþ/þ and Rab7a siRNA. PrPC and Rab7a distribution was analyzed by using 3F4 PrPC (red) and anti-Rab7a (green) antibodies. At least 25 cells were observed per condition per experiment for an equal exposure time. The scatter plots of the individual pixels from paired images. The scatter plots of the individual pixels are from paired images. The threshold levels of red on x-axis and green signals on y-axis determined the overlapping marked yellow region. Quantification of colocalization was determined by Imagej (WCIF plugin) software.

Table 2. Pearson’s Correlation of Interacting Proteins with PrPCa

A

B

coloc. coefficient

coloc. coefficient

(M1)

(M2)

treatment

PrPC

Rab7a-siRNA

PrP

þ

0.121

0.592

0.239

þ Rab7a-siRNA

þ

0.068

0.006 PrPC

0.339 Rab9

þ

0.306

0.679

0.793

þ

þ

0.779

0.970

1.000

rp

C

Rab7a

Pearson’s correlation coefficient rp (1 e rp e 1) demonstrated (A) partial co-localization in HpL3-4 PrPC knockout cells transfected with nontargeting siRNA and PrPC and (B) high co-localization between Rab9 and PrPC in HpL3-4 PrPC knockout cells transfected with Rab7asiRNA and PrPC. Co-localization coefficients, M1 and M2, ranged between 0 and 1, showed partial co-localized pixels of interest with in each channel. a

other channel.25 Results of Pearson’s correlation coefficient of colocalization demonstrated a partial colocalization between Rab7a and PrPC (Table 2, rows A). PrPC distribution was then evaluated after depleting Rab7a expression using the siRNA duplex. Approximately 7075% Rab7a expression depletion was achieved in transiently and stably PrPC expressed HpL3-4 and SH-SY5Y cells, respectively (Figure 5A). The immunoflurescence results demonstrated that a significant fraction of PrPC accumulated as a punctuated form and that the localization pattern of PrPC staining is dramatically altered in Rab7a depleted HpL3-4 cells (Figure 4D) as compared to the cells treated

similarly but without siRNA (control) (Figure 4C). Immunoblot analysis showed a significant (*P < 0.05) increase of PrPC levels in HpL3-4 cells after Rab7a knockdown in comparison to similar knockdown in PrPþ/þ control cells (without siRNA) (Figure 5A, B). These Rab7a siRNA knockdown results were confirmed in SH-SY5Y stably PrPC expressing cells (Figure 5C). The increase in the PrPC expression was confirmed in SH-SY5Y PCIneo endogenously and SH-SY5Y stable PrPC expressing cells (*P < 0.05; Figure 5A, C). To determine the subcellular localization of PrPC in these siRNA knockdown of Rab7a HpL3-4 cells, an immunofluorescence experiment with staining of PrPC and Rab9 (late endosomal marker)26 was performed. Interestingly accumulated PrPC higly colocalized with Rab9 positive compartments (Figure 6). Figure 6 showed respective scatter plots generated from representative images. Rab9 (green) and PrPC (red) was largely overlapping, as indicated in the scatter plot at 45° (Figure 6B) as compared to control HpL3-4 PrPC knockout cells transfected with non targeting siRNA and PrPC. Calculations of Pearson’s correlation coefficient of colocalization demonstrate that colocalization between PrPC and Rab9 increased after Rab7a-siRNA treatment (Table 2, rows B). To see if transiently expressed PrPC has similar characteristics to the PK resistant form, the TCL of HpL3-4 cells containing transiently transfected PrPC and treated with Rab7a-siRNA, were digested with PK (10 μg/mL) and analyzed by Western blot using SAF70 antibody. The results demonstrated that the accumulated PrPC remained sensitive to PK, at least within the tested 48 h (Figure 7).

’ DISCUSSION In the past few years, several new PrPC interacting proteins have been reported, indicating a growing interest in understanding the 3130



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Figure 5. Effect of Rab7a depletion on PrPC expression. HpL3-4 PrPC knockout with transient PrPC expressing and SH-SY5Y stable PrPC expressing cells were transfected with siRNA duplex (100 nM) to target Rab7a. (A) PrPC and Rab7a expression was analyzed after 48 H of transfection by immnoblotting using specific SAF70 PrPC and Rab7a antibodies. (B, C) Densitometry analysis from four independent ((SD) immnoblotting experiments and the significance was calculated by student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 6. Effect of Rab7a depletion on PrPC/Rab9 colocalization. HpL3-4 PrPC knockout cells transiently transfected (48 H) with PrPC and treated with siRNA (100 nM) to target Rab7a. PrPC and late endosomal marker Rab9 expression were analyzed using 3F4 anti-PrPC (red) and anti-Rab9 (green) antibodies. (A) HpL3-4 PrPC knockout cells transfected with non targeting siRNA and PrPC. (B) HpL3-4 PrPC knockout cells cotransfected with PrPC and Rab7a siRNA. At least 25 cells were observed per condition per experiment for an equal exposure time (Scale bar: 10 μm). The scatter plots of the individual pixels are from paired images. The threshold levels of red on x-axis and green signals on y-axis determined the overlapping yellow region. Quantification of colocalization was determined by Imagej (WCIF plugin) software.

physiological function of PrPC. Toward this goal, protein sequencing techniques have greatly facilitated the identification of proteins and their complexes. Although the sensitivity of mass spectrometry methods is currently sufficient to identify proteins, the isolation of protein complexes still poses serious challenges. Protein complexes need to be isolated from a densely populated cellular environment, in which the complex of interest may represent only a small fraction of the total protein population. Thus, successful purification requires a method that is stringent enough to differentiate the complex of interest from all other proteins in the mixture. On the other hand, the isolation method must also be gentle enough not to compromise the integrity of the complex. A method enabling identification of protein complexes by employing one-step purification would present several advantages. Therefore, the use of a single-step purification system known as the STrEP-tag method was explored for the isolation of interacting proteins from mammalian cells.27 To evaluate the usefulness of the STrEP-tag method for purifying protein interacting partners, mammalian expression vectors encoding PrPC fused to the One STrEP-tag at its C-terminus were first generated. The specificity of these interactions were ensured by comparative purification using control vector without the PrPC construct (PrP/). Besides reporting a number of novel PrPC interacting proteins, several already known protein partners were identified using these techniques (essentially providing a

positive control) (Table 1). Some of the previously described PrPC-interacting proteins are summarized in recent reviews.19 Interacting Partners of PrPC

Cytoskeleton-associated proteins, that is, actin, cytoplasmic 1 (known PrPSc interacting partner),28 and beta-actin-like protein 2 proteins, are involved in cell motility, cell adhesion, and reorganization of the actin cytoskeleton. PrPC also plays an important role in cell adhesion.29 Annexin A2 is known to contribute to the regulation of actin cytoskeleton dynamics in epithelial cell junctions.30 Tubulin is the major constituent of microtubules and a known interacting partner of PrPC.31 Tubulin was identified in our study as two novel isoforms, alpha-tubulin 1 and tubulin beta-5 chain. Cofilin-1 also copurified in our study although cofilin has been shown to be associated with the disease form of prion protein PrPSc and to also be involved in abnormal formation of rods in the brain of Alzheimer’s disease patients.32 Moesin, myosin-9 and vimentin have well-defined roles in the maintenance of cytoskeleton assembly.33 These cytoskeletonassociated interacting proteins are associated with PrPC during intracellular sorting and transportation.34 The 14-3-3 protein, which is involved in cell communication and signal transduction, is a biomarker for CreutzfeldtJakob Disease (CJD)35 and is also known to be an interacting partner of 3131



Figure 7. PK-digestion of PrPC in Rab7a depleted HpL3-4 cells. HpL34 PrPC knockout cells were cotransfected with C-terminus One STrEPtag PrPC and 100 nM Rab7a siRNA into HpL3-4 PrPC knockout cells. The TCL was treated with 10 μg/mL PK, and PrPC was analyzed by immunoblot using SAF70 antibody.

PrPC in association with heat shock protein 60 (HSP60).36,37 Laminin receptor 1 or 37/67 kDa laminin receptor (identified previously as an interacting partner using the yeast two hybrid system) functions as a cell surface receptor for laminin, PrPC and PrPSc. It plays a significant role in cell adhesion and in the consequent activation of signaling transduction.19,3842 Stressinduced-phosphoprotein 1 is also a known interacting partner of PrPC with a suggested role in neuroprotection.43 Ras-related protein Rab7a, ADP-ribosylation factor 1, annexin A1, A5 and endoplasmin, which were identified as novel interacting partners, have a suggested role in cell communication. Protein metabolism and energy pathways, that is, the molecular machinery required for protein metabolism, is provided by a variety of molecular chaperones that include both heat shock proteins and glucose-regulated proteins.44 Heat shock 71 kDa protein, 47 kDa heat shock protein, and stress-70 protein (GRP75) were identified as PrPC interacting partners with possible chaperone activity. Protein disulfide isomerase, which is overexpressed in the brain of sporadic CJD patients but not other neurodegenerative disorders (i.e., Down syndrome and Alzheimer’s disease), may simply reflect a cellular defense response against the altered prion protein.45 Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a known interacting partner of PrP2730 fibrils in transmissible spongiform encephalopathies (TSEs).32 Alpha enolase, a glycolytic enzyme, is upregulated in PrPC overexpressing cells in response to metabolic alterations.46 Aspartate aminotransferase plays a key role in amino acid metabolism and is important for metabolite exchange between mitochondria and the cytosol. It facilitates cellular uptake of longchain free fatty acids. It is being utilized as a cerebrospinal fluid (CSF) biomarker showing central nervous system degeneration.47 Fructose-bisphosphate aldolase A is upregulated in Scrapie-infected mice and its mRNA is increased in Scrapie infection.48 Furthermore glutathione s-transferase omega-1 is a well-known detoxification enzyme that plays an important role in prostaglandin and steroid hormone synthesis.49 It is involved in protection against oxidative stress and its isoform, glutathione s-transferase p, is reported to be up-regulated with PrPC overexpression.46 Lastly, peroxiredoxin-1, which was identified as novel interacting protein, functions to protect the ribosomal machinery against damage from oxidative stress.50 Protein involved in folding and nucleic acid metabolism is GrpE homologue 1 protein which is an essential component for the correct folding of proteins in the cell under physiological and stress conditions. It can serve as a central cellular tool for the recovery of native proteins from stress-induced aggregates. It can actively remove disease-causing toxic aggregates, such as polyglutamine-rich proteins, amyloid plaques, and prions.51 Peptidylprolyl cistrans isomerase also accelerates the folding of proteins and is involved in the protection of neurons against oxidative

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stress.52 Protein SET (Phosphatase 2A inhibitor, I2PP2A), an endogenous PP2A inhibitor, is a multitasking protein, involved in apoptosis, transcription, nucleosome assembly, and histone binding.53 Elongation factor 1-alpha, a regulator of cytoskeleton rearrangements, is upregulated in PrPC overexpressing HEK-293 cells, categorized as cell cycle and lipopolysaccharide.46 Binding immunoglobulin protein (BiP), also known as 78 kDa glucoseregulated protein, binds to a mutant prion protein for an abnormally prolonged period of time and mediates mutant prion protein degradation by the proteasomal pathway. BiP chaperoned the folding of mutant PrPC and plays a role in maintaining quality control in mutant PrPC maturation pathways.54 Oxidoreductase, stress response proteins are HSP 90-alpha and beta were up-regulated in the overexpressed PrPC conditions.46 Lactate dehydrogenase is a known interacting partner of PrPC55 and lactate dehydrogenase activity in the CSF is increased significantly in patients with CJD.56 Rab7a a Potential Partners for PrPC

The Rab-GTPases play a critical role in regulating the vesicle trafficking in both exo- and endocytic pathways.57 The importance of small GTPases in membrane trafficking is indicated by their conservation throughout eukaryotes.58,59 Our STrEP-tag affinity purification, immunofluorescence, and reverse coimmunoprecipitation results demonstrated that Rab7a (an isoform of Rab-GTPase) is potential interacting partner of PrPC. Rab7a, an important regulator of vesicular transport, is located in specific intracellular compartments (early to late endosomes) and has been shown to be involved in both the sorting and formation of transport vesicles.60 Rab7 is not only essential for the delivery of early endosome cargo to the late endosome but plays a role in biogenesis of vesicles and their fusion to the lysosome.61 Herein, new evidence is provided for Rab7a dependent mechanisms in regulating PrPC trafficking in the hippocampus neuronal cell line in association with Rab9. Intriguingly, Rab7a depletion using the siRNA knockdown system significantly increased PrPC accumulation in the cytosolic region. The localization pattern also changed to a punctuated form in contrast to the control cells. The data suggests an impairment of PrPC trafficking from early to late endosomes after knockdown of Rab7a. The immunoblot analysis in both cell lines (HpL3-4 transiently PrPC transfected and SH-SY5Y stable PrPC expressed cells) after Rab7a knockdown confirmed the increased PrPC expression. This PrPC accumulation may be attributed to the impaired biogenesis of lysosomes, which has been a strongly suggested function of Rab7a.61 Furthermore, it was demonstrated that Rab7a depletion resulted in increased PrPC accumulation and redistributed within Rab9 positive compartments. The neuropathology of most prion diseases has been accompanied by widespread deposits of amyloidal aggregates containing the misfolded prion protein or PrPSc.62 This aggregate formation has often been used as a parameter for neuronal toxicity, with characteristic resistance to PK digestion.63 However, no PK resistant PrPC was found after 48 h in siRNA transfected transiently PrPC expressed cells (Figure 7). These data suggest that impaired Rab7a machinery leads to increased accumulation of PrPC within Rab9 positive compartments but not the formation of the resistant form of PrPC at least in tested 48 h. On the basis of these results, we may speculate that PrPC can be recycled back to the plasma membrane with a Rab7a dependent pathway and that the impaired Rab7a machinery leads to increased accumulation of PrPC within Rab9 positive 3132


Journal of Proteome Research compartments. It remains to be determined whether this Rab7a dependent PrPC accumulation in the Rab9 positive endosomal compartments is crucial for the conversion of PrPC to PrPSc.

’ CONCLUSION Our proteomics approach endeavor a comprehensive list of known and novel PrPC interacting proteins. Several proteins identified in this study were unique in relation to the PrPC interaction and are crucial for various cellular pathways. Silencing of interacting protein (Rab7a) induced considerable changes in PrPC expression and localization in the neuronal cells. Furthermore, under Rab7a depleted condition PrPC, accumulation increased in Rab9 positive endosomal compartments. However, it remains to be determined whether this Rab7a-dependent PrPC accumulation in the endosomal compartments is also crucial for the conversion of PrPC in to PrPSc. These observations could also help to explain the unknown physiological role of PrPC and deserve close attention in scope of neurodegenerative diseases. ’ ASSOCIATED CONTENT

bS

Supporting Information PrPC expression in HpL3-4 cells after transient transfection (Supplementary Figure 1), PrPC localization in HpL3-4 cells after transient transfection (Supplementary Figure 2), and table showing detail MS/MS information of protein identification. This material is available free of charge via the Internet at http:// pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Dr. Abdul R. Asif, Department of Clinical Chemistry, University Medical Centre Goettingen, 37075, Goettingen, Germany. Phone: þ49-551-3922945. Fax: þ49-551-3912505. E-mail: [email protected]. )

Notes

Deceased February 27, 2010.

’ ACKNOWLEDGMENT This study was supported by a European Commission Grant (Prionscreen, FP6 - SP5A-CT-2007-044438). We are indebted to Mrs. Wiese and Mrs. Scholz for technical assistance at various stages of this investigation. We are grateful to Prof. Walson for critically reading the manuscript. Special thanks to Prof. Brose (MPI), Prof. Gross (UMG), Dr. Kirchhoff (MPI), and Dr. Bertram (IBA, Goettingen) for their support throughout the study. ’ REFERENCES (1) Le Pichon, C. E.; Valley, M. T.; Polymenidou, M.; Chesler, A. T.; Sagdullaev, B. T.; Aguzzi, A.; Firestein, S. Olfactory behavior and physiology are disrupted in prion protein knockout mice. Nat. Neurosci. 2009, 12 (1), 60–69. (2) Chen, S.; Mange, A.; Dong, L.; Lehmann, S.; Schachner, M. Prion protein as trans-interacting partner for neurons is involved in neurite outgrowth and neuronal survival. Mol. Cell. Neurosci. 2003, 22 (2), 227–233.

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