Confirmation of Prdx5 antibody ability to immunopr- 123docz.net

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 àl 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 Structurea Localization Electron

donor

Subunit References

Prdx1

Cytosol Thioredoxin GSH

Dimer

3, 4, 8 Prdx2

Cytosol Thioredoxin Dimer Decamer

Prdx3

Mitochondria Thioredoxin Dimer

Prdx4

Plasma Membrane

Thioredoxin GSH

Dimer

Prdx5

Mitochondria Peroxisome

Cytosol

Thioredoxin Monomer

Prdx6

Plasma GSH?

Cyclophillin A?

Monomer

aThe 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).

Cp Cr

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

Gene Primer name Sequence Purpose

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

β-actin Actin-F 5’-AGCGGGTCGTGCGTG-3’ RT-PCR

Actin-R 5-CAGGGTACATGGTGGTGC-3’

VEGFa

VEGFa-F1 5’-CTCACTTCCAGAAACACGACAAA-3’ Real-time VEGFa-R1 5’GCATCTTTATCTCTTTCTCTGTCATCA-3’ PCR

β-actin Actin-F1 CTGTCCACCTTCCAGCAGATGT Real-time

Actin-R1 5’-ACAGTCCGCCTAGAAGCACTTG-3’ PCR

PPIA

PPIA-F1 5’-CCCCATCTGCTCGCAATG-3’ Real-time PCR PPIA-R1 5’-GAGGAAAATATGGAACCCAAAGAA-3’

Table 3. Putative target protein altered interaction by Prdx5 immunoprecipitation under hypoxic stressa.

Accession Description Gene Score MS/MS

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

Dbt 302.6 8 ND 2/3

IPI00131695 Serum albumin Alb 325.3 3 1/3 3/3

IPI00330523 Propionyl CoA carboxylase alpha chain, mitochondrial

Pcca 474.0 21 2/3 3/3

IPI00606510 Propionyl CoA carboxylase beta chain, mitochondrial

Pccb 266.6 10 2/3 3/3

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

aProteins 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.

bFrequency 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 stressa.

Accession Description Gene Score MS/MS

spectra

Frequencyb Normoxia Hypoxia

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

IPI00130391 Protease serine 1 Prss1 1025.4 5 3/3 3/3

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

IPI00131674 Trypsinogen 2210010

C04Rik

983.5 1 2/3 2/3

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

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

H. sapiens MGLAGVCALRRSAGYILVGGAGGQSAAAAARRCSEGEWASGGVRSFSRAAAAMAPIKVGD 60 M. musculus MLQLGLRVLGCKASSVLR--ASTCLAGRAGR—-KEAGWECGGARSFSSSAVTMAPIKVGD 56 D. rerio MISTPLLQKGVRAAHCTF---RQLHCTPITSMPIKVGQ 35 D. melanogester MRVLSCKFLGRVVNSALP---QQIISLRSLSKTSAAMV--KVGD 39 * . *.: :. : ***:

H. sapiens AIPAVEVFEGEPGN--KVNLAELFKGKKGVLFGVPGAFTPGCSKTHLPGFVEQAEALKAK 118 M. musculus AIPSVEVFEGEPGK--KVNLAELFKGKKGVLFGVPGAFTPGCSKTHLPGFVEQAGALKAK 114 D. rerio RLPAVEVQEEDPGNSLSMETAELFSCKRGVLFGVPGAFTPGCSKTHLPGFIQMAGELRAK 95 D. melanogester SLPSVDLFEDSPAN--KINTGDLVNGKKVIIFGVPGAFTPGCSKTHLPGYVSSADELKSK 97 :*:*:: * .*.: .:: .:*.. *: ::******************::. * *::*

H. sapiens -GVQVVACLSVNDAFVTGEWGRAHKAEGKVRLLADPTGAFGKETDLLLDDS-LVSIFGNR 176 M. musculus -GAQVVACLSVNDVFVIEEWGRAHQAEGKVRLLADPTGAFGKATDLLLDDS-LVSLFGNR 172 D. rerio -GVDEVACISVNDVFVMSAWGKQNGADGKVRMLADPTGAFTKAVDLVLNNAQLIPVLGNL 154 D. melanogester QGVDEIVCVSVNDPFVMSAWGKEHGAAGKVRLLADPAGGFTKALDVTIDLP----PLGGV 153 *.: :.*:**** ** **: : * ****:****:*.* * *: :: . :*.

H. sapiens RLKRFSMVVQDGIVKALNVEPDGTGLTCSLAPNIISQL 214 M. musculus RLKRFSMVIDNGIVKALNVEPDGTGLTCSLAPNILSQL 210 D. rerio RSQRYAMLIENGVVTKLSVEPDGTGLTCSLASNFLAEV 192 D. melanogester RSKRYSLVVENGKVTELNVEPDGTGLSCSLANNIGKK- 190 * :*:::::::* *. *.********:**** *: :

Figure 1. Alignment of amino acid sequence of human Prdx5 and orthologues in other species. Human Prdx5 and its orthologues were applied to ClustalW 2 program (11). Amino acid numbering based on cytosolic isoform of human Prdx5 sequence. Mitochrondrial targeting sequences (MTSs) are high-lighted in gray. Peroxidatic (CP) and resolving (CR) cysteins are marked in boxes. Asterisks indicate identities, and dots and double dots indicate conservative and highly conservative substitutions, respectively. GenBank accession numbers of peptide sequences using in alignment analysis are NM_012094 (Homo sapiens), NM_012021 (Mus musculus), CN508467 (Danio rerio), and NP_650679 (Drosophila melanogaster).

152

73 CP

Mitochondrial targeting sequences

Figure 2. Immunoprecipitation using mouse anti-Prdx5 antibody. 200 àg of total lysate from mouse kidneys (input) was incubated with 5, 10, 20 àl 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 àl 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 àl antibody). Prdx5 did not exist in unbound fraction when Prdx5 antibody reached maximal concentration (20 àl 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 àl antibody (IgG concentration of Prdx5 about 5.65 àg/àl).

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).

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 àg/ml leupeptin, and 1 mM PMSF. Protein was extracted for over 4 hours at

4°C followed by sonication. Then 500 àg of protein was incubated with 10 àl Prdx5 antibody.

The co-purified proteins were pulled down by incubated with 50 àl 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.

C D

22 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 cytoskeletal derangements, reducing intracellular pH, increasing extracellular matrix production, promoting endothelial damage, stimulating macrophage infiltration and tubulointerstitial

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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Confirmation of Prdx5 antibody ability to immunoprecipitate