Identification of peroxiredoxin 5 interactome in hypoxic kidney

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.

Committee in Charge:
Park Byung-Ju

______________________________

Chay Kee-Oh

______________________________

Lee Seung-Rock

______________________________

Kim Jong-Suk

______________________________

Lee Tae-Hoon

______________________________

August 2015


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
4. Discussion................................................................................................................................22
5. References ...............................................................................................................................24
I


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

II


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

III


List of Figures

Chapter 1.

Proteomic analysis of peroxiredoxin 5 interacting proteins in hypoxic
kidney

Figure 1.

Alignment of amino acid sequence of human Prdx5 and orthologues in some
species .................................................................................................................. 16

Figure 2.

Immunoprecipitation using mouse anti-Prdx5 antibody ...................................... 17

Figure 3.

Confirmantion of VEGFa expression in hypoxia treated kidney via semiquantified 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

IV

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

V


List of Tables

Chapter 1.

Proteomic analysis of peroxiredoxin 5 interacting proteins in hypoxic
kidney

Table 1.

Schematic representation of mammalian peroxiredoxin family members. .......... 11

Table 2.

The list of primers used for RT-PCR and Realtime-PCR .................................... 12

Table 3.

Putative target protein showing altered interaction by Prdx5
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

VI


List of Abbreviation
AEBSF

4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride

Ala

Alanine

Alb

Albumin

Arg

Arginine

ATP

Adenosine triphosphate

BCA

Bicinchoninic acid

BCAAs

Branched chain amino acids

BCKDH

Brached chain alpha keto-acid dehydrogenase

Cys

Cysteine

DBT

Dihydrolipoamide branched chain transacylase E2

DTT

Dithiothreitol

ECM

Extracellular matrix

EDTA

Ethylenediaminetetraacetic acid

EGTA

Ethylene glycol tetraacetic acid

E.coli

Escherichia coli

Gba2

Glucosidase, beta (bile acid) 2

Gln

Glutamine

Glu

Glutamic acid

GSH

Glutathione

GTP

Guanosine-5'-triphosphate

HA

Human influenza hemagglutinin

HEK

Human embryonic kidney

HIF

Hypoxia-inducible factor

HRP

Horseradish peroxidase

IAM

Iodoacetamide
VII


Ile

Isoleucine

IPTG

Isopropyl β-D-1-thiogalactopyranoside

Krt

Keratin

Leu

Leucine

NAD+/ NADH

Oxidized /reduced form of nicotinamide adenine dinucleotide

PBS

Phosphate buffered saline

PCC

Propionyl-CoA carboxylase

Pcca

Propionyl-CoA carboxylase, alpha polypeptide

Pccb

Propionyl-CoA carboxylase, beta polypeptide

PCR

Polymerase chain reaction

PMSF

Phenylmethylsulfonyl fluoride

PPIA

Peptidylprolyl isomerase A

Prdx

Peroxiredoxin

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

Thr

Threonine

Txn1

Thioredoxin 1
VIII


UPLC-MS/MS

Ultra performance liquid chromatography tandem mass
spectrometry

UUO

Unilateral ureteral obstruction

Val

Valine

VEGF

Vascular endothelial growth factor

VHL

Von Hippel-Lindau

IX


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


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
cytosol and mitochondria, even in some situations existing in nucleus (38). Moreover, human
Prdx5 existing in mitochondria just share 28% to 30% sequence identity with human typical 22


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
3


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.

4


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 RNAsefree water and kept in 4oC.
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
5


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
EDTA and 2% (w/v) SDS in 0.1 M tetra ethyl ammonium bicarbonate (TEABC) and incubated
at 37 °C for 30 min for complete dissolution. Proteins were reduced by adding 10 µl of 20 mM
6


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.
7


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 HRPconjugated secondary antibody (Cell Signaling Technology, USA) for analysis.

8


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
9


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.

10


Table 1.

Schematic representation of mammalian peroxiredoxin family members.
Structurea

Name

Localization

Electron

Subunit

References

donor
Cp

Cytosol

Cr

Prdx1

Thioredoxin

Dimer

GSH

Cp

Cytosol

Cr

Thioredoxin

Prdx2

Dimer
Decamer

Cp

Cr

Cp

Cr

Mitochondria

Thioredoxin

Dimer

Plasma

Thioredoxin

Dimer

Membrane

GSH

Mitochondria

Thioredoxin

Monomer

GSH?

Monomer

Prdx3

Prdx4

Cp

Prdx5

Cr

3, 4, 8

Peroxisome
Cytosol
Plasma

Cp

Prdx6

Cyclophillin
A?

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).

11


Table 2.
Gene
VEGFa

β-actin

VEGFa
β-actin

PPIA

The list of primers used for RT-PCR and Realtime-PCR.
Primer name

Sequence

Purpose

VEGFa-F

5’-ACATCTTCAAGCCGTCCTGTGTGC-3’

RT-PCR

VEGFa-R

5’-AAATGGCGAATCCAGTCCCACGAG-3’

Actin-F

5’-AGCGGGTCGTGCGTG-3’

Actin-R

5-CAGGGTACATGGTGGTGC-3’

VEGFa-F1

5’-CTCACTTCCAGAAACACGACAAA-3’

Real-time

VEGFa-R1

5’GCATCTTTATCTCTTTCTCTGTCATCA-3’

PCR

Actin-F1

CTGTCCACCTTCCAGCAGATGT

Real-time

Actin-R1

5’-ACAGTCCGCCTAGAAGCACTTG-3’

PCR

PPIA-F1

5’-CCCCATCTGCTCGCAATG-3’

Real-time

PPIA-R1

5’-GAGGAAAATATGGAACCCAAAGAA-3’

PCR

12

RT-PCR


Table 3.

Putative target protein altered interaction by Prdx5 immunoprecipitation
under hypoxic stressa.

Accession

Description

Gene

Score

MS/MS

Frequencyb

spectra

Normoxia

Hypoxia

IPI00130467

Ras related protein Rab 43 isoform b

Rab43

228.7

6

ND

1/3

IPI00130535

Lipoamide acyltransferase component of

Dbt

302.6

8

ND

2/3

branched chain α-keto acid
dehydrogenase complex, mitochondrial
IPI00131695

Serum albumin

Alb

325.3

3

1/3

3/3

IPI00330523

Propionyl CoA carboxylase alpha chain,

Pcca

474.0

21

2/3

3/3

Pccb

266.6

10

2/3

3/3

mitochondrial
IPI00606510

Propionyl CoA carboxylase beta chain,
mitochondrial

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

IPI00226993

Thioredoxin

Txn1

1155.1

3

3/3

2/3

IPI00225123

Non-lysosomal glucosylceramidase

Gba2

191.4

9

1/3

ND

IPI00348328

Keratin Kb40

Krt78

580.5

19

1/3

ND

IPI00122281

Keratin type I cuticular Ha2

Krt32

260.3

8

1/3

ND

13


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.

14