Identification of peroxiredoxin 5 interactome in hypoxic kidney

Proteomic analysis of peroxiredoxin 5 interacting proteins in hypoxic kidney Tran Gia-Buu Department of Molecular Medicine Graduate School, Chonnam National University Supervised by Pr

Doctoral Dissertation

Identification of peroxiredoxin 5 interactome in hypoxic kidney

Department of Molecular Medicine Graduate School, Chonnam National University

Tran Giabuu

August 2015

Identification of peroxiredoxin 5

interactome in hypoxic kidney

Department of Molecular Medicine Graduate School, Chonnam National University

Tran Gia-Buu Supervised by Professor Lee Tae-Hoon

The thesis entitled above, by the graduate student named above, in

partial fulfillment of the requirements for the Doctor of Philosophy in

Science has been deemed acceptable by the individuals below

Contents Contents I List of Figures IV List of Tables VI List of Abbreviation VII

Chapter 1 Proteomic analysis of peroxiredoxin 5 interacting proteins in hypoxic

kidney

Abstract (in English) 1

1 Introduction 2

2 Materials and methods 5

1) Hypoxic treatment 5

2) Extraction of total RNA 5

3) Reverse-transcription polymerase chain reaction 5

4) Producing Prdx5 antibody 6

5) Protein extraction and immunoprecipitation 6

6) Interactome analysis by nano UPLC-MS/MS 6

7) Confirmation of Prdx5 interacting protein via western blot analysis 8

3 Results 9

1) Confirmation of Prdx5 antibody ability to immunoprecipitate 9

2) Confirmation of hypoxic stress in mouse kidney 9

3) The short-list of putative proteins altered in hypoxic kidneys 9

4) Confirmation of the data collected from LC-MS/MS analysis by reverse immunoprecipitation 10

Abstract (in Korean) 29

Chapter 2 Interaction between peroxiredoxin 5 and dihydrolipoamide branched chain transacylase E2 under hypoxic condition Abstract (in English) 30

1 Introduction 31

2 Materials and methods 34

1) Reagents 34

2) Hypoxic treatment 34

3) Protein extraction and immunoprecipitation 34

4) Determination of DBT enzymactic activity 34

5) Cell culture and plasmid construction 35

6) Confocal fluorescence microscopy 35

7) Analysis of the role of Prdx5 cysteine residues in interaction between Prdx5 and DBT in hypoxic stress 36

3 Results 37

1) Analysis of Prdx5 and DBT interaction under hypoxic stress 37

2) The effect of hypoxic stress on DBT enzymatic activity 37

3) Confirmation of DBT overexpressing construct 37

4) The co-localization of Prdx5 and DBT under hypoxic stress 38

5) The role of Prdx5 cysteine residues in the Prdx5-DBT interaction 38

4 Discussion 51

5 References 54

Abstract (in Korean) 57

Chapter 3 Primary evaluation of interaction between Prdx5 and Alb, Rab43, Pcca

and Pccb

Abstract (in English) 58

1 Introduction 59

2 Materials and methods 61

1) Vibrio vulnificus-infected mouse model 61

2) Staphylococcus aureus-infected cell model 61

3) Protein extraction and immunoprecipitation 62

4) Pcca and Pccb overexpressing vector construction 62

5) Escherichia coli culture and IPTG induction 62

6) Protein purification 62

3 Results 64

1) Interaction between Prdx5 and Alb in human serum albumin administered Vibrio vulnificus-infected mouse model 64

2) Expression of Prdx5 and Rab43 in Staphylococcus aureus-infected macrophages 64

3) Interaction between Prdx5 and Rab43 in Staphylococcus aureus-infected cell model 64

4) Confirmation of Pcca and Pccb relating constructs 64

5) Examination of solubility of mature Pcca and Pccb 64

6) Optimization of Pcca and Pccb induction 65

7) Examination of the ability of Pcca and Pccb to produce PCC complex in vitro 65

4 Discussion 82

5 References 84

Abstract (in Korean) 87

quantified RT-PCR and realtime-PCR 18 Figure 4 Work-flow to identify putative target protein interacted with Prdx5 in

hypoxic kidney 19 Figure 5 Confirmation of putative target proteins interacted with Prdx5 by reverse

immunoprecipitation 21

Chapter 2 Interaction between peroxiredoxin 5 and dihydrolipoamide branched

chain transacylase E2 under hypoxic condition

Figure 1 Scheme of function of BCKDH complex in BCAAs catabolic pathway 40 Figure 2 Coprecipitation of endogenous Prdx5 with DBT in normoxic and hypoxic

mouse kidney 42 Figure 3 In vitro assay of DBT enzymatic activity in normoxic and hypoxic mouse

kidneys 43 Figure 4 Cloning DBT/pCMV construct 44

Figure 5 Confirmation of WT and mutant Prdx5 constructs 47 Figure 6 Co-localization of Prdx5 and DBT in normoxic and hypoxic HEK293

cells 49 Figure 7 Comparative interactions of Prdx5 WT or cysteine mutants with DBT in

normoxic and hypoxic cells 50

Chapter 3 Primary evaluation of interaction between Prdx5 and Alb, Rab43, Pcca

and Pccb

Figure 1 Schematic representation to generate V vulnificus-infected mouse model 67

Figure 2 Interaction between Prdx5 and albumin in the spleens and livers collected from V vulnificus-infected mice 68

Figure 3 Expression of Prdx5 and Rab43 in S aureus-infected macrophages 69

Figure 4 Interaction between Prdx5 and Rab43 in S aureus-infected macrophages 71

Figure 5 Confirmation of Pcca and Pccb overexpressing vectors 72

Figure 6 Examination of solubility of Pcca and Pccb 75

Figure 7 Optimization IPTG induction to improve Pccb solubility 76

Figure 8 Purification of Pcca and Pccb 78

Figure 9 Examination of the ability of Pcca and Pccb to generate PCC complex 80

immunoprecipitation under hypoxic stress 13 Table 4 List of proteins interacted with Prdx5 is not altered during hypoxic stress 15

Chapter 3 Primary evaluation of interaction between Prdx5 and Alb, Rab43, Pcca

and Pccb

Table 1 The list of primers used for cloning and sequencing Pcca and Pccb

constructs 74

ATP Adenosine triphosphate

BCAAs Branched chain amino acids

BCKDH Brached chain alpha keto-acid dehydrogenase

DBT Dihydrolipoamide branched chain transacylase E2

ECM Extracellular matrix

EDTA Ethylenediaminetetraacetic acid

EGTA Ethylene glycol tetraacetic acid

E.coli Escherichia coli

Gba2 Glucosidase, beta (bile acid) 2

GTP Guanosine-5'-triphosphate

HA Human influenza hemagglutinin

HIF Hypoxia-inducible factor

Pcca Propionyl-CoA carboxylase, alpha polypeptide

Pccb Propionyl-CoA carboxylase, beta polypeptide

PCR Polymerase chain reaction

PMSF Phenylmethylsulfonyl fluoride

PPIA Peptidylprolyl isomerase A

PVDF Polyvinylidene difluoride

Rab43 Ras-related protein Rab43

RIPA Radioimmunoprecipitation assay

ROS/RNS Reactive oxygen species/ reactive nitrogen species

RT-PCR Reverse-transcription polymerase chain reaction

SDS Sodium dodecyl sulfate

SPF Specific-pathogen free

TCEP Tris-2-carboxyethyl phosphine

TEABC Tetra ethyl ammonium bicarbonate

TEMED Tetramethylethylenediamine

TFA Trifluoroacetic acid

UPLC-MS/MS Ultra performance liquid chromatography tandem mass

spectrometry UUO Unilateral ureteral obstruction

VEGF Vascular endothelial growth factor

Proteomic analysis of peroxiredoxin 5 interacting proteins in

hypoxic kidney

Tran Gia-Buu

Department of Molecular Medicine Graduate School, Chonnam National University (Supervised by Professor Lee Tae-Hoon)

(Abstract)

Peroxiredoxin 5 (Prdx5) plays a major role in preventing oxidative damage as an effective antioxidant protein within variety cells through peroxidase activity However, the function of Prdx5 is not only limited to peroxidase enzymatic activity It also appears to have unique function in regulating cellular response to external stimuli by directing interaction with signaling protein In this study, imunoprecipitation coupled with nano-UPLC-MSE shotgun proteomics was employed to identify putative interacting partners of Prdx5 in mouse kidney during hypoxia A total of 17 proteins were identified as potential interacting partners of Prdx5

by a comparative interactomic analysis in kidney between normoxia and hypoxia These results will contribute to enhance the understanding of Prdx5’s role in hypoxic stress and may suggest new directions for future research

1 Introduction

Peroxiredoxin (Prdx, formerly named as TSA and TPx) is a family of thiol-dependent peroxidase, which has ability to reduce hydrogen peroxide, alkyl hydroperoxides, peroxynitrite and thereby plays major roles in preventing oxidative damage through their peroxidase activity

as well as mediates signal transduction (1-2) Peroxiredoxins ubiquitously express in organisms from all kingdoms with a variety cellular localizations They have fast reactivity with hydrogen peroxide (∼107

M−1s−1) implies in mammalian cells (39-40) Prdx family members are distributed in variety of subcellular location such as cytosol, mitochondria, peroxisome and plasma (3-4) Recently, the studies suggest that Prdxs also serve divergent functions related in various biological processes such as the cell proliferation, differentiation and several genes expression (5-7)

Six isoforms of mammalian Prdxs (Prdx1-6) were characterized and classified into three sub-groups basing on resolution mechanism and the existence or the lack of a resolving cysteine (Cr) localized to the C-terminal region of the enzyme: 1-Cys, typical 2-Cys, and atypical 2-Cys The 1-Cys peroxiredoxin subfamily (Prdx6) possess only one conserved peroxidase cysteine (Cp) in the N-terminus whereas typical 2-Cys peroxiredoxin subfamily (Prdx1-4) contains both the N- and C-terminal-conserved Cys (Cp and Cr) residues and require both of them for catalytic function In contrast, atypical 2-Cys subfamily (Prdx5) contains only the N-terminal conserved Cp but require one additional, less conserved Cys residue for catalytic activity (8) All Prdxs share the same basic catalytic mechanism, in which a peroxidatic cysteine

is oxidized to a sulfenic acid by the peroxide substrate The recycling of the sulfenic acid back to

a thiol is what distinguishes the three enzyme classes In 1-Cys Prdx, the sulfenic acid formed during Cp oxidation is reduced by an external thiol, whereas in typical 2-Cys-Prdxs and atypical 2-Cys-Prdx, the Cr of one subunit attacks sulfenic acid of a second subunit resulting in the formation of a stable inter-molecular disulfide bond or intra-molecular disulfide bond, respectively (Table 1)

Among 6 isoforms, Prdx5 is the last identified one and the only one member of atypical 2-Cys-Prdx subfamily Human Prdx5 was described firstly in 1999 as a DNA-binding protein potentially implicated in the repression of RNA-polymerase-III–driven transcription of the Alu-family retroposons and later as thioredoxin peroxidase (9-10) Unlike other Prdx members, Prdx5 addresses surprisingly wide intracellular localization from peroxisomes, to

Cys and 1-Cys peroxiredoxins suggesting human Prdx5 as the divergent member of Prdxs family (11) Prdx5 is a monomeric protein and posses a conserved peroxiase Cys residue at position 48 (Cp) and two additional Cys residues at positions 73 and 152 (Cr) (Figure 1) Mutational analyses indicate that Cys48 is a catalytic site which transiently forms an intramolecular disulfide with Cys152 during the catalytic cycle This mechanism distinguishes Prdx5 from intermolecular disulfide formation in typical 2-Cys Prdxs members (12) Cytoprotective antioxidant function of mammalian Prdx5 was investigated in a variety of cell line and tissue (13-16) Furthermore, Prdx5 also contains an N-terminal mitochondrial targeting sequence and SQL (Ser–Gln–Leu) peroxisomal targeting sequence type 1 at its C-terminus, indicating Prdx5

as an effective peroxidase for peroxisomes and mitochondria, two organelles that are major intracellular sources of ROS/RNS This protein appears to be multifunctional, and the full spectrum of cellular functions of Prdx5 remains unknown Recently, Prdx5 was reported to be a stress-inducible factor under oxidative stress, especially in hypoxic stress (17-19)

Hypoxia is one of the most important factors influencing in the pathogenesis and progression of acute and chronic renal disease (20) Although kidney is supplied a high overall oxygen, the parallel arrangement of arterial and venous preglomerular and postglomerular vessels just allows oxygen to pass through via shunt diffusion Thus, the partial pressure oxygen

of tissue in kidney, especially in renal medulla, is comparatively low (oxygen tension < 10 mmHg) Furthermore, kidney is second organ only to the heart in terms of O2 consumption to maintain active transtubular re-absorption of solutes, in particular sodium The high demand of oxygen combines with insufficient low oxygen pressure rendering kidney to be particularly susceptible to hypoxic damage (21) Relationship between hypoxia and progression of renal disease can be demonstrated in 3 main points: the chronic renal diseases are associated with a rapid reduce in capillary density; consequently, it make declined oxygen delivery to tubular cells, and the partial pressure of oxygen in renal tissue usually reduce during renal diseases, the inadequately low oxygen tensions, in its turn, could regulate cellular functions via specific stimulating certain genes such as hypoxia-inducible factor (HIFs) system (22) A body of evidence has accumulated to suggest the link between HIFs target genes and renal diseases At

first, Higgin and collaborators showed inhibition HIF-1a could ameliorate the development of tubulointerstitial fibrosis in UUO (unilateral ureteral obstruction) kidneys (23) Second, Rankin

and collaborators found that conditional inactivation of VHL, von Hippel-Lindau tumor

suppressor, the protein that regulates the protein stability of HIF-alpha, in PEPCK-Cre mutants resulted in renal cyst development and inactivation HIF1β suppressed cystic formation providing

the role of HIFs system in VHL-associated renal disease (24) It is well known that kidney could alter expression of antioxidant enzymes to prevent hypoxic injury (Cu/Zn-SOD, GSH reductase, catalase, Mn-SOD) (25-26) However, the role of peroxireodoxin family in renal hypoxic

response, especially Prdx5, have not elucidated yet Recently, Yang and collaborators reported

that Prdx5 exerted protective effects in hypoxic kidney by regulating a variety of individual proteins in a set of protein network (27)

To gain further insights into the mechanisms regulated by Prdx5 in hypoxic condition,

I employed an approach for comparing the interacted partners in kidneys under normoxia versus hypoxia Here, I suggested Prdx5 interactome using the strategy of immunoprecipitation complex in hypoxic kidney These data will reveal the interaction between putative proteins and Prdx5 in hypoxic kidney and provide better understanding about metabolic homeostasis in hypoxic kidney

2 Materials and methods

1) Hypoxic treatment

Mice (C57BL/6J) were maintained under specific-pathogen free (SPF) conditions All animal-related procedures were reviewed and approved under the Animal Care Regulations (ACR) of Chonnam National University (accession number: CNU IACUC-YB-2013-39)

To produce hypoxic condition, a chamber was designed to regulate the flow of N2 using a gas supply and the oxygen concentration in chamber was monitored and maintained at 8.0±0.5% O2 during experiments using an oxygen controller (Proox Model 110; BioSpherix, USA) After 4 hours of hypoxia, all mice (N=3/each group, 8 weeks-aged) were induced with anesthesia under hypoxic condition and kidneys were rapidly removed and frozen in liquid N2 Hypoxic condition was determined according to previous study (27)

2) Extraction of total RNA

Mouse kidneys were ground in liquid nitrogen and subsequently homogenized 100 mg

of tissues in Qiazol reagent (Qiagen, Netherlands) Total RNA was purified following the manufacturer's instruction Extracted total RNA was treated with DNase I (Takara, Shiga, Japan)

to remove genomic DNA contamination, and then phenol-chloroform extraction was performed

to stop the reaction The quality and concentration total of RNA were determined at absorbance

of 260 nm as well as the ratio 260/280 nm, 260/230 nm by spectrophotometry (ND-2000, Nano Drop Technologies, USA)

3) Reverse-transcription polymerase chain reaction

For preparing cDNA, 1 μg of total RNA was reverse-transcribed using PrimeScript RT

reagent kit (Takara, Shiga, Japan) which utilized random hexamers and oligo dT in the reverse

transcription reaction After reverse-transcription, the products were diluted 5 folds in free water and kept in 4oC

RNAse-For the semi-quantitative PCR amplification, 1 μ1 of cDNA (<200 ng) was used as a template in a 20-μl final reaction volume PCR amplification was accomplished using the following condition: 25 cycles at 94oC for 30 sec, 55 oC for 30 sec, 72 oC for 1 min, followed by

a final elongation step at 72 oC for 5 min Expression pattern of VEGFa and β-actin (reference

gene) were analyzed by 2% agarose gel electrophoresis and visualized with ethidium bromide under UV illumination (UVP GELDOC-It TS Imaging System, USA)

For quantification of mRNA expression of VEGFa of mouse kidney, real-time PCR

analysis was performed for the using a 7300 real-time PCR system (Applied Biosystems, USA) according to the manufacturer’s instructions Samples were amplified with SYBR premix Ex

Taq (Takara, Shiga, Japan) Reactions were analyzed in triplicate β-actin and PPIA were used as

reference genes Relative quantification of mRNA expression was performed using the

2−ΔΔCTmethod The primer sets for the PCR analysis of the expression patterns in kidneys are listed in Table 2

4) Producing Prdx5 antibody

The purified mouse Prdx5 protein (2.5 mg of protein per rabbit) from bacterial induction system was coupled to 10 mg of keyhole limpet hemocyanin (Thermo scientific, Rockford, USA) by incubation overnight at room temperature in the presence of 7 mM glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.0) The protein-hemocyanin conjugate was mixed with incomplete Freund’s adjuvant (Sigma-Aldrich, USA) for the initial injection and with complete Freund’s adjuvant (Sigma-Aldrich, USA) for booster injections After the initial injection of 1 mg of peptide, rabbits were subjected to three booster injections, using 500 µg of protein per injection, administering (at multiple subcutaneous sites) in 4-week intervals Blood was collected at 1 week after the third booster injection, and the antisera were extracted and used

in following experiment

5) Protein extraction and immunoprecipitation

For protein extraction, hypoxic mouse kidneys were homogenized in a lysis buffer containing 1% Triton X-100 in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM sodium orthovanadate, 25 mM sodium fluoride, 1 µg/ml leupeptin, and 1 mM PMSF Protein was extracted by sonication The cleared extract was collected by centrifugation at 13,000 rpm for 30 min at 4 °C The protein concentration in the cleared extract was measured using a BCA protein assay (Pierce Biotechnology, Rockford, USA)

For analysis of proteins interacting with Prdx5, 500 µg of protein was incubated with

10 μl of Prdx5 antibody at 4 °C for overnight The immune complex was pulled down by incubating with protein G agarose (Invitrogen, Carlsbad, USA) for 4 hours at 4 °C The immunoprecipitated complex was eluted with 60 mM Tris-HCl (pH 6.8), 2.5% glycerol, 2% SDS, and 28.8 mM β-mercaptoethanol and then the eluted complex was freeze-dried before being subjected to nano-UPLC-MS/MS analysis for comparative proteomics

6) Interactome analysis by nano-UPLC-MS/MS

For gel-assisted digestion, the dried pellet was resuspended in 50 μl of 6 M urea, 5 mM

tris-2-carboxyethyl phosphine (TCEP) and alkylated by adding 20 µl of 20 mM iodoacetamide (IAM) at room temperature for 30 min To incorporate proteins into a gel directly in the Eppendorf vial, 18.5 μl of acrylamide/bisacrylamide solution (40%, v/v, 29:1), 2.5 μl of 10% (w/v) ammonium persulfate, and 1 μl of 100% TEMED was then applied to the protein solution The gel was cut into small pieces and then washed three times with three volumes of TEABC containing 50% (v/v) ACN The dehydrated gel samples were then digested with 15 µl trypsin (0.1 µg/ µl) at 37 oC for 18 hours Then the digested peptides were recovered twice with a solution containing 50 mM ammonium bicarbonate, 50% acetonitrile, and 5% trifluoroacetic acid (TFA) The resulting peptide extracts were pooled, dried in a vacuum centrifuge, and then

dissolved in 0.1% formic acid solution prior to MS/MS analysis

For nano-LC and tandem MS analysis, a nano-ACQUITY Ultra Performance LC Chromatography™ equipped Synapt™ G2-S System (Waters Corporation, MA, USA) used was previously described (41) This step was performed on a 75 μm × 250 mm nano-ACQUITY UPLC 1.7 μm BEH300 C18 RP column and a 180 μm × 20 mm Symmetry C18 RP 5 μm enrichment column using a nano-ACQUITY Ultra Performance LC Chromatography™ System (Waters Corporation, MA, USA) Trypsinized peptides (5 μl) were loaded onto the enrichment column in mobile phase A (3% acetonitrile in water with 0.1% formic acid) A step gradient was then used at a flow rate of 300 nl/min This included 3–40% mobile phase B (97% acetonitrile in water with 0.1% formic acid) run over 95 min, followed by 40–70% mobile phase B run over 20 min, and finally a sharp increase to 80% B over 10 min Sodium formate (1 μmol/min) was used

to calibrate the TOF analyzer in the range of m/z 50–2000, and [Glu1]-fibrinopeptide (m/z 785.8426) was run at 600 nL/min for lock mass correction During data acquisition, the collision energies of low-energy mode (MS) and high-energy mode (MSE) were set to 4 eV and 15–40 eV energy ramping, respectively One cycle of the MS and MSE modes of acquisition was performed every 3.2 s In each cycle, MS spectra were acquired for 1.5 s with a 0.1 s interscan delay (m/z 300–1990), and the MSE fragmentation (m/z 50–2000) data were collected in triplicate

The continuum LC-MSE data were processed and searched using the IDENTITYE algorithm in PLGS (ProteinLynx GlobalServer) version 2.5.2 (Waters Corporation, USA) The data acquired by alternating low and high energy modes in the LC-MSE were automatically smoothed, background subtracted, centered, deisotoped and charge state reduced, after which alignment of the precursor and fragmentation data were combined with retention time tolerance (± 0.05 min) using PLGS software

Processed ions were mapped against the IPI mouse database (version 3.87) using the following parameters: peptide tolerance, 10 ppm; fragment tolerance, 0.05 Da; missed cleavage, 1; and carbamidomethylation at C and oxidation at methionine and cysteine Peptide identification was performed using the trypsin digestion rule with one missed cleavage As a result, protein identification was completed with arrangement of at least two peptides All proteins identified on the basis the IDENTITYE algorithm are in keeping with > 95% probability The false positive rate for protein identification was set at 5% in the databank search query option, based on the automatically generated reversed database in PLGS 2.5.2 Protein identification was also based on the assignment of at least two peptides comprised of seven fragments or more

7) Confirmation of Prdx5 interacting proteins via western blot analysis

To confirm the interaction between Prdx5 and the candidates, the immunoprecipitated complexes from normoxic and hypoxic mouse kidney were purified by antibodies were specific for target candidates such as anti–DBT antibody (#ab59746, Abcam, USA), anti-Rab43 antibody (#sc-100113, Santa Cruz, USA), anti-Alb antibody (sc58688, Santa Cruz, USA), and anti-Pccb antibody (H00005096-D01, Abnova, USA) The purified immunoprecipitates were separated on 15% SDS-PAGE gel and transferred onto PVDF membrane (Bio-Rad Laboratories, USA) The membranes were incubated with anti-Prdx5 (1:5000), anti-Alb (1:1000), anti-Rab43 (1:200) and anti-Pccb (1:1000) as the primary antibody and then with HRP-conjugated secondary antibody (Cell Signaling Technology, USA) To detect DBT from immunoprecipitated complex, the membranes were incubated with anti-DBT (1:2000) overnight at 4oC then with anti-mouse Ig light chain antibody (#AP200P, Millipore, USA) The membranes were next probed with HRP-conjugated secondary antibody (Cell Signaling Technology, USA) for analysis

3 Results

1) Confirmation of Prdx5 antibody ability to immunoprecipitate

Neither commercial nor laboratory made mouse Prdx5 antibody had not been tested in immunoprecipitate assay yet It is necessary to verify whether the Prdx5 antibody interacted with Prdx5 protein or not In briefly, Prdx5 antibody produced in my laboratory and carried out immunoprecipitation with mouse kidney lysate Western blot analysis carried with commercial anti-Prdx5 antibody under manufacturer instruction (#LF-PA0010, LabFrontier, Korea) Prdx5

in unbound part was decreased whereas Prdx5 immunoprecipitated in bound part was increased depending on Prdx5 antibody concentration (Figure 2) Prdx5 did not exist in unbound fraction when Prdx5 antibody reached maximal concentration (20 µl antibody), that indicated Prdx5 was pulled down into bound fraction completely

2) Confirmation of hypoxic treatment in mouse kidney

To confirm hypoxic condition was successfully induced in mouse kidney, I used

VEGFa as hypoxic indicator in these studies RT-PCR and realtime-PCR results showed that VEGFa expression was induced in hypoxic kidney This result is lower than another research

group (VEGFa expression was upregulated about twice in hypoxic group), but the difference in

VEGFa expression between hypoxic and normoxic groups was significant in these experiments

(1.05±0.15 and 1.40±0.12, normoxia versus hypoxia, the results were presented in SEM±STD and using β-actin as reference gene) The difference results between two groups can be explained by the different hypoxic treatment (O2 concentration and time course) In this study, I used 8.0±0.5 % O2 during 4 hours whereas the previous group used 6% O2 during 6 hours to

induce hypoxic stress (28) VEGFa is a well know indicator for hypoxic stress, thus the upregulation of VEGFa in hypoxic mouse kidneys sample proves this condition is enough to

induce hypoxic stress in mouse kidney (Figure 3)

3) The short-list of putative proteins in hypoxic kidneys

To investigate the putative target interacting with Prdx5 in hypoxic condition, I initially exposed normal mice (C57BL6/J) to hypoxia (8.0±0.5% O2 for 4 hours), then the mice were sacrificed and mouse kidneys were collected for next experiment The mouse kidney lysate was applied to immunoprecipitate with Prdx5 antibody that previously confirmed the co-purify

ability According to Han and collaborators, to maximize protein digestion efficiency and

recovery (>90%), I employed the gel-assisted digestion method (29) I next subjected the digested protein to a nano-UPLC-MSE proteomic analysis to identify proteins interacting with Prdx5 I compared the proteomic data from three independent experiments to determine

meaningful targets with high reproducibility (Figure 4) In detail, 27 (149 spectra) and 33 (276 spectra) proteins were identified as Prdx5 interaction proteins under normoxic and hypoxic condition, respectively Table 3 summarizes the potential interacting partners of Prdx5 under hypoxia condition Among them, 13 proteins increased interaction with Prdx5 in the hypoxic

versus the normoxic kidney: Rab43, DBT, Alb, Pcca, Krt76, Krt14, Krt17, Krt84, Krt72, Krt74,

Krt77, Krt42, and Pccb On the other hand, 4 proteins showed decreased interaction with Prdx5

in the hypoxic versus the normoxic kidney: Gba2, Txn1, Krt78, and Krt32 (Table 3)

Furthermore, some proteins did not show the change of interaction with Prdx5 under hypoxic

conditions: Prss1, Hbb-b1, 2210010C04Rik, Krt1, Krt71, Krt2, Krt18 (Table 4)

4) Confirmation of the data collected from LC-MS/MS analysis by reverse immunoprecipitation

To confirm my proteomics analysis for identifying Prdx5 interacting partners, coprecipitation experiments were performed with some representative proteins As shown in Figure 5, DBT, Rab43, Alb, and Pccb were shown to strongly coprecipitate with Prdx5 in hypoxia, consistent with the proteomics analysis in Table 3 Taken together, these findings suggested that Prdx5 could act as a direct regulator in hypoxia and be involved in maintaining kidney homeostasis

Table 1 Schematic representation of mammalian peroxiredoxin family members

Name Structure a Localization Electron

Thioredoxin GSH

Dimer

Prdx5

Mitochondria Peroxisome Cytosol

Thioredoxin Monomer

Prdx6

Cyclophillin A?

Monomer

a

The cysteins that relate with peroxidase activity are indicated as Cp (peroxidase cystein) or Cr (resolving cystein) Prdx3 and Prdx5 have mitochondrial import signals at their N-terminal regions, beside that Prdx5 also has a peroxisomal localization signal at its C-terminus Prdx4 has

a signal peptide for secretion at the N-terminus (8) Prdx5 exists in ubiquitous cell line and appears to be multifunctional, in some case it plays a role as a stress-inducible factor under specialized oxidative stress conditions, especially hypoxic stress (42)

Table 2 The list of primers used for RT-PCR and Realtime-PCR

VEGFa VEGFa-F 5’-ACATCTTCAAGCCGTCCTGTGTGC-3’ RT-PCR

Table 3 Putative target protein altered interaction by Prdx5 immunoprecipitation

under hypoxic stress a

spectra

Frequencyb Normoxia Hypoxia

IPI00130467 Ras related protein Rab 43 isoform b Rab43 228.7 6 ND 1/3

IPI00130535 Lipoamide acyltransferase component of

branched chain α-keto acid dehydrogenase complex, mitochondrial

IPI00330523 Propionyl CoA carboxylase alpha chain,

IPI00346834 Keratin type II cytoskeletal 2, oral Krt76 184.2 7 ND 1/3

IPI00227140 Keratin type I cytoskeletal 14 Krt14 1399.7 16 ND 1/3

IPI00230365 Keratin type I cytoskeletal 17 Krt17 1322.1 16 ND 1/3

IPI00347019 Keratin type II cuticular Hb4 Krt84 330.8 10 ND 1/3

IPI00347096 Keratin type II cytoskeletal 72 Krt72 337.2 8 ND 1/3

IPI00462140 Keratin type II cytoskeletal 1b Krt77 2014.2 7 ND 1/3

IPI00468696 Keratin type I cytoskeletal 42 Krt42 798.6 12 ND 1/3

IPI00420970 Keratin type II cytoskeletal 74 Krt74 1999.7 4 1/3 3/3

Proteins were affinity-purified from mouse kidneys under both normoxic and hypoxic conditions as bound interactors with Prdx5 immunoprecipitation The purified immunoprecipitates were applied to acrylamide gel-associated tryptic digestion and subjected to nano-UPLC-MS/MS for protein identification

b

Frequency represents the number of times that the interactors were observed in three independent experiments ND, not detected

Table 4 List of proteins interacted with Prdx5 is not altered during hypoxic stress

spectra

Frequencyb Normoxia Hypoxia

IPI00625729 Keratin type II cytoskeletal 1 Krt1 2912.3 7 3/3 3/3

IPI00988950 Hemoglobin subunit beta 1 like Hbb-b1 1493.7 6 2/3 2/3

C04Rik

IPI00468956 Keratin type II cytoskeletal 71 Krt71 2022.0 4 1/3 1/3

IPI00622240 Keratin type II cytoskeletal 2 epidermal Krt2 657.2 8 1/3 1/3

IPI00311493 Keratin type I cytoskeletal 18 Krt18 297.0 8 1/3 1/3

a

Proteins were affinity-purified from mouse kidneys under both normoxic and hypoxic conditions as bound interactors with Prdx5 immunoprecipitation The purified immunoprecipitates were applied to acrylamide gel-associated tryptic digestion and subjected to nano-UPLC-MS/MS for protein identification

b

Frequency represents the number of times that the interactors were observed in three independent experiments ND, not detected

sequences using in alignment analysis are NM_012094 (Homo sapiens), NM_012021 (Mus

musculus), CN508467 (Danio rerio), and NP_650679 (Drosophila melanogaster)

Figure 2 Immunoprecipitation using mouse anti-Prdx5 antibody 200 µg of total lysate from

mouse kidneys (input) was incubated with 5, 10, 20 µl of mouse Prdx5 antibody at 4 oC in circulator for overnight The immune complex was pulled down by incubating with protein G agarose (Invitrogen, USA) for 4 hours at 4 °C Unbound and bound fractions were separated by centrifugation at 3000 rpm, followed by washing several times with 1x PBS After that, unbound and bound fractions were applied into SDS-PAGE and western blot with commercial Prdx5 antibody The results from western blotting showed that Prdx5 existed in bound fraction from start point at 5 µl antibody Additionally, the decrease of Prdx5 amount in unbound fraction got along with the increase of Prdx5 amount in bound fraction when I added more Prdx5 antibody (5, 10, 20 µl antibody) Prdx5 did not exist in unbound fraction when Prdx5 antibody reached maximal concentration (20 µl antibody), that indicated Prdx5 was fully pulled down into bound fraction Taken together, these results indicated Prdx5 antibody produced in my laboratory could co-purified mouse Prdx5 and the concentration using for immunoprecipitation assay in a range 5-20 µl antibody (IgG concentration of Prdx5 about 5.65 µg/µl)

Figure 3 Confirmation of VEGFa expression in hypoxia treated kidney via semi-quantified

RT-PCR and realtime-PCR Mouse kidneys from hypoxic and normoxic groups were

homogenized in Qiazol under manufacturer’s instructions They were divided in two groups, one group (n=3) used for RT-PCR (A) and one group (n=3) used for realtime-PCR (B) The results

from RT-PCR showed upregulation of VEGFa during hypoxic treatment, 1.30±0.10 versus

0.97±0.06 (hypoxia versus normoxia group, respectively Relative expressions were normalized

with β-actin and represented in mean ± standard deviation, p<0.05 Additionally, relative expression of VEGFa also increased in hypoxic group, 1.40±0.12 (β-actin) and 1.38±0.07

(PPIA) compared with normoxic group, 1.05±0.15 (β-actin) and 1.01±0.03 (PPIA)

A

B

Figure 4 Work-flow to identify putative target protein interacting with Prdx5 in hypoxic kidney C57BL6/J mice were divided into two groups, one group maintained in normoxia

(20.0±0.5% O2 ) whereas other maintained in O2 concentration regulated chamber ( 8.0±0.5% O2, for 4 hours) After indicated time point, the mice were sacrificed and collected the kidneys I prepared the total lysate from normoxic and hypoxic kidneys in a lysis buffer containing 1% Triton X-100 in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM sodium orthovanadate, 25 mM sodium fluoride, 1 µg/ml leupeptin, and 1 mM PMSF Protein was extracted for over 4 hours at

4°C followed by sonication Then 500 µg of protein was incubated with 10 µl Prdx5 antibody The co-purified proteins were pulled down by incubated with 50 µl protein G (50% slurry) The immunoprecipitated complex was eluted with 60 mM Tris-HCl (pH 6.8), 2.5% glycerol, 2% SDS, and 28.8 mM β-mercaptoethanol and then the eluted complex was freeze-dried before being subjected to gel associated trypsin digestion and nano-UPLC-MS/MS analysis for comparative proteomics Processed ions were mapped against the IPI mouse database (version 3.87) for identify putative protein

Figure 5 Confirmation putative target proteins interacting with Prdx5 by reverse immunoprecipitation To confirm my proteomics analysis for identifying Prdx5 interacting

partners, I carried out reverse immunoprecipitation assay with target protein [(A) Pccb, (B) Alb, (C) Rab43, (D) DBT] The data indicated that all target proteins strongly co-precipitated with Prdx5 in hypoxic condition, these results also were consistent with my data collected from nano-

UPLC-MS/MS comparative analysis

A

B

4 Discussion

Prdx5 displaying remarkably wide subcellular distribution compares with the other mammalian peroxiredoxins Prdx5 is a peroxidase that utilizes cytosolic thioredoxin 1 or mitochondrial thioredoxin 2 to reduce alkyl hydroperoxides or peroxynitrite with high rate constants, and Prdx5 response to hydrogen peroxide is slower than other Prdxs (30-31) Although Prdx5 appeared to be constitutively and ubiquitously expressed in most mammalian tissues, its expression is upregulated in various pathophysiologic situations and in response to various kinds of stresses (32-33) These characteristics suggest that this protein may have unique functions, which can be differentiated from other Prdx isoforms, in mammalian cells

In this study, I have improved a manageable and rapid protocol for purification and identification of mouse Prdx5 and its interacting proteins in hypoxic kidney through direct immunoprecipitation of Prdx5 followed by shotgun proteomic analysis Using this approach, I identified novel interacting partners of Prdx5 from three independent replicates To assure the result of Prdx5 interacting partners from my proteomic analysis, coimmunoprecipitation was conducted by using anti-target protein antibodies in normoxic and hypoxic mouse kidneys, and my candidate targets has been successfully validated On the other hand, how the hypoxia regulating Prdx5 and its partners interaction has unknown yet To investigate that mechanism requires more researches characterize the effect of hypoxia on Prdx5 and its interacting protein’s structure as well as their physiological functions

Because oxygen tension in renal medulla is very low ~10 mmHg and kidney requires high consumption oxygen for activating many physiological processes such as the sodium transport system, oxidative phosphorylation and the synthesis of ATP, it is well known that the kidney is prone to acute hypoxic injury (21) Moreover, hypoxic stress can promote kidney injury via altering kidney energy metabolism by regulating HIF, the well- known transcription factor that regulate the expression of glucose transporters or activating the lipid peroxidation (22) During hypoxic stress, oxygen deficiency prevents the use of BCAAs as the mitochondrial electron transfer system for production of ATP, as the consequence, BCAAs accumulate in plasma (34) Although BCAAs are essential amino acids, the accumulation of BCAAs and their metabolites can toxic to the cells, thus hypoxia-inducing BCAAs accumulation may be promotes kidney injury On the other hand, hypoxia also contributes to renal damage by

fibrosis, transdifferentiation of tubule cells to myoblasts Kidney also has some mechanisms to maintain homeostasis during hypoxic stress such as upregulation of antioxidant enzymes (25-26)

Recently, Yang and collaborators found that knocking down Prdx5 influenced the

expression of a variety of protein group associated with oxidative stress, mitochondrial transport, fatty acid metabolism, amino acid/nucleic acid metabolism, glycolysis/gluconeogenesis, and cytoskeleton In addition, hypoxic kidney in Prdx5 knock-down mice (Prdx5si) showed insufficient activity of mitochondrial metabolic enzymes, especially aconitase 2 (Aco2), acyl-CoA dehydrogenase C-4 to C-12 straight chain (Acadm), and acyl-CoA oxidase 1 (Acox1) (27) Taken together, Prdx5 may be involved in the coupling of a broad range of cellular signaling cascades to maintain renal homeostasis under hypoxic conditions In these studies, from functional annotation analysis, Prdx5 interacting partners such as DBT, Pcca, Pccb, Gba2 appeared to be related to various metabolisms associated with mitochondrial localization For example, DBT (dihydrolipoamide branched chain transacylase E2) is second component of branched-chain α-keto acid dehydrogenase (BCKDH) complex which involved in the breakdown of the branched-chain amino acids (BCAA), such as isoleucine, leucine, and

valine The mutation in DBT gene will cause accumulation of BCAAs and its toxic metabolites,

manifested in patients with maple syrup urine disease (35) Addionally, Pcca (propionyl coA carboxylase, alpha polypeptide) and Pccb (propionyl coA carboxylase, beta polypeptide) are two component of propionyl coA carboxylase, the enzyme converts propionyl CoA to methylmalonyl CoA, thus mutation of Pcca or Pccb related with propionic acidemia, a kind of autosomal recessive metabolic disease, in which the accumulation of dangerous acids and toxins occurs and causes damage to the organs (36) Gba2 (non-lysosomal glucosylceramidase) is an enzyme that catalyzes the conversion of glucosylceramide to free glucose and ceramide and the hydrolysis of bile acid 3-O-glucosides Gba2 was related to carbohydrate transport and metabolism, but recent study also suggested its function in motor neuron defects of hereditary spastic patients (37) As my knowledge, this study is the first study to identified interaction between Prdx5 and DBT, Pcca, Pccb, Gba2 and it provides the strong clue to prove that Prdx5 can directly interact with metabolic related proteins and maintain cellular homeostasis in hypoxic kidneys Taken together, these data suggest that Prdx5 may be related with metabolic pathway and it is a promising target for treatment hypoxic related diseases

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