Proteomic and Phosphoproteomic Analysis at Diapause Initiation in

Proteomic and Phosphoproteomic Analysis at Diapause Initiation in the Cotton Bollworm, Helicoverpa armigera Yu-Xuan Lu and Wei-Hua Xu* State Key Laboratory of Biocontrol and School of Life Sciences, Sun Yat-Sen (Zhongshan) University, Guangzhou 510275, China Received April 21, 2010

Diapause is a period of developmental arrest that allows a species to adapt to unfavorable conditions. Many insect species reduce metabolic activity and then enter diapause at a certain stage in their life cycles. The cotton bollworm, Helicoverpa armigera, will be destined for pupal diapause when larvae are reared under short daylengths and low temperature. The brain is an important organ for diapause decision, and some signaling molecules from the brain of diapause-destined individuals are released into the hemolymph to regulate diapause. In this study, we performed 2-D gel-based comparative proteomic and phosphoproteomic analyses to search for differentially expressed proteins between nondiapause- and diapause-destined pupal brains. A total of 79 proteins and 23 phosphoproteins showed significant differences between these two groups, and 41 proteins and 10 phosphoproteins were identified by MALDI-TOF/TOF MS. Further, gene expression patterns in diapause- and nondiapausedestined pupal brains were confirmed by RT-PCR or Western blot analysis. These differentially expressed proteins act in the metabolic change, stress response, and signal transduction pathways at early pupal stage for diapause initiation. Thus, these identified proteins may depress metabolism in diapausedestined pupae to lead the insect to enter developmental arrest. Keywords: diapause • proteome • phosphoproteome • 2-DE • Helicoverpa armigera

1. Introduction A distinct feature of insect life cycles is their physiological and behavioral adaptations to ubiquitous, seasonally changing environments. To enhance survival during unfavorable periods, many species undergo a state of developmental arrest called diapause, which is characterized by greatly reduced metabolic activity and extreme stress resistance. Diapause is a dynamic and complicated developmental process not only in insects, but also in other invertebrates and vertebrates, such as designated dauer in nematode Caenorhabditis elegans and dormancy (or hibernation) in the ground squirrel Spermophilus tridecemlineatus, respectively.1,2 Diapause is usually induced by environmental signals and occurs at a specific stage for each species. In the cotton bollworm, Helicoverpa armigera, which is an important agricultural noctuid moth, pupal diapause is induced by incubating larvae at low temperature and short day lengths (20 °C, 10 h light/day), and pupae will enter diapause 10 days after pupation. If larvae are incubated at high temperature and long day lengths (25 °C, 14 h light/day), all pupae develop toward adults. A developmental or diapause program will be released from the brain and promotes a distinct set of genes that regulate development or diapause.3 A classical theory for pupal diapause is that diapause initiation is likely to be a failure of prothoracicotropic hormone (PTTH) synthesis * To whom correspondence should be addressed. Wei-Hua Xu, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China. Tel: +86 20 39332967. Fax: +86 20 84112297. E-mail: [email protected]. 10.1021/pr100356t

 2010 American Chemical Society

and release from the brain, and the lack of PTTH results in a failure to stimulate the prothoracic gland to produce the ecdysteroids needed to promote pupal-adult development.2,3 Ecdysteroids are a class of the most important insect hormones that regulate the expression of a series of cascaded genes for embryogenesis, growth, development, differentiation, and reproduction.4 However, little is known about what differentially expressed genes regulate developmental arrest at early pupal stage (diapause initiation phase). It is usually thought that far fewer genes are expressed during diapause, and diapause does indeed represent a shutdown in gene expression.5 By using pulse labeling and 2-dimensional electrophoresis (2-DE), Joplin et al. observed that the protein synthesis ratio was reduced in the diapausing pupal brain of the flesh fly Sarcophaga crassipalpis.6 Flannagan et al. found that slightly fewer diapause-related genes were up-regulated as compared to down-regulated in pupal brain of S. crassipalpis by using elimination hybridization.7 A similar result was also found in a recent proteomic study in S. crassipalpis pupal brain.8 This evidence shows that diapause may be a unique developmental pathway instead of a simple shutdown in gene expression. Recently, proteomic analysis is regarded as a powerful approach for large-scale investigations of gene expression at the protein level.9 Many proteomic studies on insect development and metamorphosis have been reported,10-14 but the identified proteins from insect diapause are scarce.8,15 In addition to protein expression, protein phosphorylation, which Journal of Proteome Research 2010, 9, 5053–5064 5053 Published on Web 08/05/2010

research articles is the best known as post-translation modification (PTM) of target proteins, also plays a key role in development through reverse phosphorylation on serine/threonine and tyrosine amino groups on target proteins.16 The changed protein phosphorylation level is likely to be a quick response to metabolic changes from development to diapause. Thus, a combination of proteomic and phosphoproteomic analyses is a useful strategy for studying the molecular mechanism of insect diapause. In this study, 2-DE is coupled with silver staining for total proteomic analysis, and Pro-Q diamond staining for phosphoproteomic analysis and mass spectrometry analysis. Differentially expressed proteins are compared between diapauseand nondiapause-destined pupal brains. In total, 41 proteins and 10 phosphoproteins are identified at diapause initiation. Further, gene expression patterns in the two types of pupae are investigated at the mRNA and protein levels. The results show that changes of protein and protein phosphorylation are involved in the regulation of diapause initiation in the early pupal stage.

2. Materials and Methods 2.1. Animals. When larvae of H. armigera were individually reared on an artificial diet at 25 °C and a light-dark cycle of L14:D10 h, all pupae developed without entering diapause. When larvae were reared at 20 °C and a light-dark cycle of L10:D14 h, over 90% of the pupae entered diapause. Pupal brains were dissected in an insect saline containing 0.75% NaCl and stored at -80 °C until use. 2.2. Separation of the Brain Proteins by 2-DE and Image Analysis. Pupal brains were homogenized in 250 µL of lysis buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, and 40 mM dithiothreitol (DTT)), precipitated in 10% trichloroacetic acid (TCA)/acetone, centrifuged at 12 000g at 4 °C, and then washed twice with ice-cold acetone. The proteins were dissolved in rehydration buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 40 mM DTT, 0.5% (v/v) IPG buffer 3-10NL (GE Healthcare), and 0.001% (w/v) bromophenol blue). The protein concentration was determined by Bradford’s method as described previously.17 The sample proteins (600 µg each) were used to rehydrate 18 cm IPG strips (pH 3-10 NL) (GE Healthcare) overnight, with 350 µL of rehydration buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 40 mM DTT, 0.5% (v/v) IPG buffer (pH 3-10NL), and 0.001% (w/v) bromophenol blue). Isoelectric focusing (IEF) was carried out at 20 °C in an IPGphor II apparatus (GE Healthcare) under the gradient procedures as: 250 V for 1 h, 500 V for 1 h, 1000 V for 2 h, gradient to 8000 V for 1 h, and 8000 V for 5 h. Then, the strips were soaked for 15 min in an equilibration buffer (6 M urea, 30% (v/v) glycerol, 2% SDS, 50 mM Tris-HCl (pH 8.8), and 1% DTT) and then for additional 15 min in a modified equilibration buffer (DTT was replaced with 2.5% iodoacetamide). The second dimension was performed using 12% SDS polyacrylamide gels. Gels were stained with Pro-Q Diamond phosphoprotein gel stain (Molecular Probes, Invitrogen) by fixing in 50% ethanol/10% acetic acid overnight, washing three times (15 min each) in deionized water, incubating in Pro-Q Diamond phosphoprotein gel stain for 120 min in the dark, destaining with three washes of 20% acetonitrile in 50 mM sodium acetate (pH 4.0), and using two additional washes in deionized water. Images were acquired with Typhoon Variable Model Images Trio+ (GE Healthcare) using 532 nm excitation and 580 nm bandpass emission filter. After fluores5054

Journal of Proteome Research • Vol. 9, No. 10, 2010

Lu and Xu cent images were acquired, the gels were stained for total proteins with silver stain according to the method described by Yan et al.18 The stained gels were scanned at 300 dpi, using Image Scanner III (GE Healthcare). Then, the scanned gels were analyzed automatically using ImageMaster 2D software (version 7.0). Protein spots displaying g1.5 average-fold increase or decrease in abundance (p-value < 0.05) were selected for protein identification. 2.3. In-Gel Digestion and MS. Silver-stained protein spots were excised from the gel and digested in gel as reported in Shevchenko et al.19Briefly, the gel particles were washed in deionized water twice (10 min each), placed in 100% CH3CN, and then dried in a speed vacuum. Dried gel pieces were covered with 10 µL of 12.5 ng/µL sequencing grade trypsin (Promega) in 25 mM NH4HCO3 buffer. In-gel digestion was incubated at 37 °C overnight. Each 2.5-µL sample was spotted on an AnchorChip plate (Bruker Daltonics) followed by 1 µL of 0.4 mg/mL HCCA in 70% acetonitrile and 0.1% TFA. Samples were analyzed using Ultraflex III TOF/TOF mass spectrometer (Bruker Daltonics). External calibration was performed using Bruker peptide calibration standards. Mass spectra (MH+) were acquired by FlexControl (version 3.0, Bruker Daltonics) which recorded in the range 800-4 500 Da, and the MS/MS information were obtained in LIFT (laser-induced forward transfer) mode. 2.4. Database Construct and Search. An in-house database was constructed with the EST sequences from our H. armigera EST library (unpublished data). MS and MS/MS spectra were combined by BioTools software (version 3.1, Bruker Daltonics) and searched against our in-house database using MASCOT software (Matrix science). The search parameters were set as (100 ppm for PMF peptide tolerance and (0.6 Da for the MS/ MS tolerance. The combined spectra were also searched against the NCBInr database or NCBI EST_others database (taxonomy of Metazoan) to obtain more information. The search results from the combined spectra that were statistically significant (p < 0.05) were accepted. 2.5. Bioinformatics Analysis. Functional annotation of the identified proteins was performed using Blast2GO.20 The identified protein sequences were submitted to Blast2GO to obtain gene ontologies that were based on Gene Ontology (GO) terms (www.geneontology.org). 2.6. RT-PCR Analysis. Total RNA was extracted form H. armigera brain using the single-step method of acid guanidinium thiocyanate-phenol-chloroform extraction.21 Total RNA (1 µg) was used for first-strand cDNA synthesis using M-MLV reverse transcriptase (Promega). The gene-specific primers used in PCR are listed in Supplementary Table 1, and actin was used as internal control. The PCR reactions were subjected to 25-30 cycles consisting of 94 °C, 30 s; 56 °C, 30 s; 72 °C, 30 s. 2.7. Western Blot analysis. A total of 15 µg of total protein from the brains was lysed in lysis buffer (150 mM NaCl, 1.0% Triton X-100, 50 mM Tris (pH 8.0), 1 mM PMSF, 1 mM EGTA, 5 mM NaF, and 10 mM Na3VO4), separated on a 12% SDS-PAGE gel, transferred to PVDF membranes (0.4 µm, Millipore), immunoblotted with anti-MnSOD serum (diluted 1:3000) and anti-ArgK serum (diluted 1:2000), and visualized by enhanced chemiluminescence (Super Signal West Pico, Thermo Scientific). For the global phosphotyrosine profile analysis, 30 µg of total proteins was run on 6-12% gradient SDS-PAGE gel and incubated with anti-phosphotyrosine ((4G10 Platinum, Millipore), 1:1000).

Proteome and Phosphoproteome at Diapause Initiation

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Figure 1. 2-DE gel images of the total proteome in H. armigera pupal brain. The proteins were separated by IEF and SDS-PAGE and stained with MS-compatible silver stain. (A) Day 1 nondiapause pupae (NP1); (B) day 2 diapause type pupae (DP2); (C) day 2 nondiapause pupae (NP2); and (D) day 4 diapause type pupae (DP4). The differentially expressed protein spots were marked with a letter and number; letters ‘a’ and ‘b’ represent (DP2/NP1) and (DP4/NP2), respectively. The protein spots, which were observed in all three biological replicates, were excised and analyzed by MALDI-TOF/TOF.

3. Results 3.1. Proteome and Phosphoproteome Patterns of Pupal Brain. To identify differentially expressed proteins at early pupal stage of diapause initiation, we carried out 2-DE analysis to compare the total proteome and phosphoproteome of nondiapause- and diapause-destined pupal brains. The nondiapause- and diapause-destined pupae were, respectively, incubated at 25 and 20 °C; thus, their rates of development differed significantly. On the basis of preliminary experiments, the pupal stage lasted for 12 or 25 days when the pupae were incubated at 25 or 20 °C, respectively. Meanwhile, we considered that diapause initiation stage in H. armigera is from day 0 to day 9 after pupation. Thus, two developmental points were selected to identify differentially expressed proteins. The first time point compares the day 1 pupal brain of nondiapause pupae (NP1) with the day 2 pupal brain of diapause pupae (DP2). The other time point compares day 2 pupal brain of nondiapause pupae (NP2) with the day 4 pupal brain of diapause pupae (DP4).

In total proteome, 776, 758, 782, and 775 protein spots were detected in NP1, DP2, NP2, and DP4, respectively (Figure 1). Of these proteins, 79 differentially expressed spots were found in all three biological replicates, including 42 up-regulated spots and 37 down-regulated spots in diapause pupae (Figure 3A). In the phosphoproteomic analysis, 138 (NP1), 105 (DP2), 176 (NP2), and 129 (DP4) protein spots were detected (Figure 2). Of these proteins, 23 spots were differentially expressed phosphoproteins in all three biological replicates, including 8 upregulated spots and 15 down-regulated spots in diapause pupae (Figure 3B). 3.2. Identification and Annotation of Differentially Expressed Proteins. The isolated protein spots were subjected to in-gel digestion and MALDI-TOF/TOF analysis. Up to now, there was no H. armigera genomic sequence or H. armigera EST database deposited in GenBank or other data library, so we constructed two EST libraries from H. armigera brain and fat body, which contains 3586 genes or unique sequence. First, the protein MS/MS data were submitted to the in-house H. Journal of Proteome Research • Vol. 9, No. 10, 2010 5055

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Figure 2. 2-DE gel images of the phosphoproteome in H. armigera pupal brain. The phosphoproteins were separated by IEF and SDSPAGE and stained with Pro-Q Diamond stain. (A) Day 1 nondiapause pupae (NP1); (B) day 2 diapause type pupae (DP2); (C) day 2 nondiapause pupae (NP2); and (D) day 4 diapause type pupae (DP4). The differentially expressed phosphoprotein spots were marked with a letter and number; letters ‘pa’ and ‘pb’ represent (DP2/NP1) and (DP4/NP2), respectively. The phosphoprotein spots, which were observed in all three biological replicates, were excised and analyzed by MALDI-TOF/TOF.

armigera EST database (EST_Har). Twenty-five spots in the total proteome and 4 spots in the phosphoproteome were identified because our EST database could not cover all H. armigera proteins to obtain more information for these protein spots. Then, the spectra were also submitted to the NCBInr database and the EST_others database. An additional 16 spots in the total proteome and 6 spots in the phosphoproteome were identified during this search. Finally, we identified 41 proteins from 79 spots (Table 1; Figure 4) and 10 proteins from 23 phosphoprotein spots (Table 2). Of these proteins, 22 proteins are more abundant and 19 proteins are less abundant in the total proteome at diapause initiation. In addition, three phosphoproteins are more abundant and seven phosphoproteins are less abundant in the phosphoproteome. Surprisingly, more than one different spots identified as the same protein, such as actin depolymerizing factor 1 (ADF1) (Table 1 and Figure 1, a4 and b2, b3), bombyrin (Table 1 and Figure 1, a5, a6 and b4, b5), heat shock protein 20.7 (Hsp20.7) (Table 1 and Figure 1, b44, b47), and vesicle amine transport protein (Table 1 and Figure 5056


1, b15, b16). It may be caused by post-translational modification or protein degradation. In addition, the identified proteins and phosphoproteins were annotated by GO analysis. In the proteome, metabolic process was the top biological process both in high-abundance proteins (30%) and low-abundance proteins (23%) (Supplementary Figure 1A,B). In the phosphoproteome, metabolic process and cellular process were the two major biological processes in both high- and low-abundance phosphoproteins (Supplementary Figure 1C,D). 3.3. Confirmation of Differentially Expressed Proteins. To confirm the differential expression at diapause initiation, mRNAs that encode five high-abundance proteins (acetoacetylCoA thiolase (ACAT), bombyrin, fructose 1,6-bisphosphate aldolase (aldolase), heat shock protein 21.4 (Hsp21.4), and malate dehydrogenase (MADH)) and four low-abundance proteins (ADF1, calmodulin, elongation factor 1 beta’ (EF1beta’) and reptin) were detected in pupal brain by RT-PCR (Figure 5A). The results showed that transcription levels of eight


research articles and 8 high- and 15 low-abundance phosphoproteins were isolated from diapause type brain. Combining MALDI-TOF/ TOF and database searches, 41 proteins in the proteome and 10 proteins in the phosphoproteome were identified. Further, gene expressions were checked by RT-PCR at the mRNA level and Western blot at the protein level, and the differential expression was confirmed as identified in 2-DE analysis. These differentially expressed proteins and phosphoproteins in diapause-destined pupal brain may result in diapause initiation in early pupal stage. 4.1. Metabolic Changes. At the early stage of diapausedestined pupae, development starts to slow down toward diapause that accompanies metabolic depression. We identified 41 differentially expressed proteins and found that the major biological process altered in diapause-destined pupa is metabolic changes: 30% of these proteins are more abundant, and 23% of these proteins less abundant, at diapause initiation. Six energy metabolism-related proteins are aldolase, MADH, 6-phosphogluconolactonase, 3-hydroxybutyrate dehydrogenase type 2, acetoacetyl-CoA thiolase, and ArgK, and nearly all of these proteins are more abundant in diapause-destined pupal brain, with the exception of ArgK.

Figure 3. Statistics of differentially expressed proteins (A) and phosphoproteins (B) during (DP2/NP1) and (DP4/NP2). The spots are up-regulated or down-regulated g1.5-fold (p < 0.05) in diapause pupae as compared to the nondiapause pupae.

genes are consistent with the protein levels identified in proteome, the exception is EF-1beta’ (Figure 5B and C). With antibodies specifically recognizing H. armigera MnSOD and arginine kinase (ArgK) made by our laboratory, we investigated the presence of MnSOD and ArgK in diapause- and nondiapause-destined pupal brains using Western blot. The results were also consistent with the 2-DE analysis that these two proteins did not change in DP2 but decreased in DP4 (Figure 5D). 3.4. Tyrosine Phosphorylation Analysis. High molecular (>100 kDa) proteins are hard to display in 2-DE gels.22,23 To further investigate the change of high molecular proteins in total phosphorylation level, proteins from pupal brain were detected using a phosphotyrosine antibody, which is highly specific compared to antibodies against phosphoserine and phosphothreonine.24 The tyrosine phosphorylation level in NP1 was a little higher than in DP2, but it increased significantly in NP2 compared to DP4. The result further confirms that global protein phosphorylation increases significantly in nondiapause pupae but not in diapause pupae (Figure 5E). These results also implicate that tyrosine kinases act in certain proteins of nondiapause individuals for their development, and a low level in tyrosine phosphorylation may induce diapause initiation in the early pupal stage.

4. Discussion The insect brain is the center of developmental control where some signaling molecules are generated and released into the hemolymph to regulate development or diapause.25 In this study, the total number of 1533 protein spots expressed in diapause-destined brains is similar to nondiapause-destined brains (1558 spots) at early pupal stage, and 42 high- and 37 low-abundance proteins were found. The number of phosphoprotein spots in diapause-destined brains (234 spots) is far fewer than that in nondiapause-destined brains (314 spots),

Three enzymes, aldolase, MADH, and 6-phosphogluconolactonase, participate in glucose metabolism. Aldolase catalyzes the fourth step in glycolysis, which cleaves fructose 1,6bisphosphate and generates dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. MADH is the last reaction enzyme of the TCA cycle (tricarboxylic acid cycle) and catalyzes malate into oxaloacetate by producing NADH, which was up-regulated at the mRNA level in the northern house mosquito diapause adult, Culex pipiens.26 In addition, 6-phosphogluconolactonase is a core component of the pentose phosphate pathway (PPP), which produces NADPH. The expression of these three enzymes increases synchronously at diapause initiation. On the basis of metabolic pathway shown in Figure 6, we conclude that these three enzymes may be involved in the biosynthesis of polyols (e.g., glycerol and sorbitol) at diapause initiation. Aldolase may participate in glycerol biosynthesis as an antifreeze agent that protects the insect from coldness,27 and MADH and PPP produce NADH and NADPH involved in glycerol and sorbitol. The polyols are widespread in diapause insects not only as good cryoprotectants, but also as development arrester by reducing the free water content to repress enzymes activity.28 Sorbitol and trehalose have been demonstrated predominant in diapausing pupae in H. armigera,29 and glycerol concentration maintains a high level throughout diapause stage in S. crassipalpis.30 Similarly, in the diapause egg of silkworm B. mori, glycogen converts to glycerol and sorbitol at diapause initiation and reverses this process when diapause is terminated.31,32 Thus, the up-regulation of polyols by aldolase, MADH, and PPP at the early pupal stage may play two key roles. One is to promote diapause initiation through depressing metabolic activity, and the other is to induce cryoprotectant biosynthesis for cold hardiness in diapause individuals. Lipid is heavily synthesized and stored in the fat body of diapause-destined larvae as the predominant energy reserves during diapause period.33 For example, the respiratory quotient (RQ) values from the solitary bee Megachile rotundata is near 0.7 during the first 3 months of overwintering, indicating that lipid reserves serve as the primary fuel resource at the early diapause stage.34 The lipid in fat body can be broken into ketone bodies (e.g., R-β-hydroxybutyrate) and then transported Journal of Proteome Research • Vol. 9, No. 10, 2010 5057

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Table 1. Identification of Differentially Expressed Proteins spot no.

ratio

accession no.

description

theoretical (pI/kDa)

no. of peptides matched

Mowse scorea

EST_Har EST_Har

3 4

208/29 203/32

EST_Har

1

89/30

EST_Har

3

61/29

EST_Har NCBInr

2 1

155/29 72/46

NCBInr

1

64/46

EST_Har

2

166/28

EST_others

1

58/57

NCBInr

2

63/46

EST_others

1

57/57

EST_Har

3

186/30

EST_Har EST_Har

1 2

93/26 92/30

EST_Har

3

54/32

EST_Har

2

148/30

EST_Har EST_others

1 1

92/31 80/57

EST_others

3

158/57

EST_others

1

92/57

EST_Har

1

41/28

EST_others

2

91/57

EST_Har EST_Har

1 4

45/32 115/30

EST_others

2

113/58

NCBInr

3

188/46

EST_Har

1

99/30

EST_Har

2

65/30

NCBInr

3

82/46

EST_Har

2

73/30

EST_others

3

135/56

EST_others

2

70/57

database

Proteins in DP2/NP1 a5 a4

4.59 3.84

gi|112983654 gi|153792659

a13

2.97

gi|193664723

a16

2.85

gi|91077926

a6 a21

2.82 2.09

gi|112983654 gi|114052166

a19

2

gi|66508517

a11

1.96

gi|112983414

a24

1.75

gi|114051966

a20

1.63

gi|66516056

a22

1.62

gi|17137546

a10

2.73

gi|160947856

a1 a30

2.58 2.44

gi|17564542 gi|113208389

a31

1.53

gi|114052396

Proteins More Abundant in Diapause bombyrin [Bombyx mori] 7.05/22.54 actin-depolymerizing factor 6.17/17.23 1 [B. mori] similar to glutathione 5.85/23.44 S-transferase-like protein [Acyrthosiphon pisum] similar to 3-hydroxybutyrate 9.05/27.20 dehydrogenase type 2 [Tribolium castaneum] bombyrin [B. mori] 7.05/22.54 UDP-galactose 4-epimerase 5.87/43.13 [B. mori] similar to F-actin capping 5.37/32.95 protein subunit alpha [Apis mellifera] heat shock protein 5.79/21.39 hsp21.4 [B. mori] mitochondrial aldehyde 5.57/52.76 dehydrogenase [B. mori] similar to Serine/threonine-protein 5.85/38.32 phosphatase PP1-beta catalytic subunit [A. mellifera] flotillin, isoform A 5.56/47.14 [Drosophila melanogaster] Proteins Less Abundant in Diapause elongation factor 1 beta’ 4.53/24.40 [Spodoptera exigua] calmodulin [D. melanogaster] 4.09/16.81 heat shock protein 20.7 6.23/20.75 [Mamestra brassicae] ubiquitin-conjugating enzyme E2M 6.83/20.90 [B. mori] Proteins in DP4/NP2

b2

special

gi|153792659

b4 b32

5.53 2.23

gi|112983654 gi|170063044

b37

2.13

gi|148298685

b31

2.12

gi|237874151

b36

2.06

gi|153791621

b40

1.85

gi|17136898

b5 b39

1.83 1.71

gi|112983654 gi|91077926

b7

1.64

gi|148298750

b42

1.60

gi|14517793

b27

special

gi|170050075

b47

4.67

gi|113208389

b16

4.30

gi|153792203

b44

2.76

gi|113208389

b29

2.40

gi|170048590

b12

2.11

gi|66532600

5058

Proteins More Abundant in Diapause actin-depolymerizing 6.17/17.23 factor 1 [B. mori] bombyrin [B. mori] 7.05/22.54 malate dehydrogenase [Culex 6.35/38.73 quinquefasciatus] fructose 1,6-bisphosphate 8.38/39.65 aldolase [B. mori] glutamine synthetase 6.03/41.48 2 [A. pisum] acetoacetyl-CoA thiolase 7.56/39.61 [B. mori] walrus, isoform B 8.39/34.18 [D. melanogaster] bombyrin [B. mori] 7.05/22.54 similar to 3-hydroxybutyrate 9.05/27.20 dehydrogenase type 2 [T. castaneum] 6-phosphogluconolactonase 5.41/25.95 [B. mori] glutathione-S-transferase-like 6.91/24.31 protein [Galleria mellonella] Proteins less abundant in diapause glycyl-tRNA synthetase [C. 7.79/83.22 quinquefasciatus] heat shock protein 20.7 6.23/20.75 [M. brassicae] vesicle amine transport 5.85/49.13 protein [B. mori] heat shock protein 20.7 6.23/20.75 [M. brassicae] T-complex protein 6.17/59.64 1 subunit gamma [C. quinquefasciatus] similar to phospholipase 5.67/86.58 A2, activating protein [A. mellifera]


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Proteome and Phosphoproteome at Diapause Initiation Table 1. Continued spot no.

ratio

accession no.

b43

2.05

gi|112983802

b46 b20

1.99 1.90

gi|24646906 gi|17737543

b15

1.88

gi|153792203

b17

1.81

gi|112983388

b45

1.77

gi|114052396

b3

1.66

gi|153792659

b19

1.61

gi|17737635

b21

1.57

gi|156968301

a


Mowse scorea

EST_Har

1

29/29

6.81/16.90 5.67/47.03

EST_Har EST_Har

1 3

82/29 114/31

5.85/49.13

NCBInr

4

179/46

8.00/51.02

EST_others

1

106/57

6.83/20.90

EST_Har

2

41/30

6.17/17.23

EST_Har

2

185/32

5.63/53.91

EST_Har

2

129/30

5.76/40.32

EST_Har

2

78/30

description


Mn superoxide dismutase [B. mori] effete [D. melanogaster] actin-related protein 66B [D. melanogaster] vesicle amine transport protein [B. mori] FK506-binding protein FKBP59 homologue [B. mori] ubiquitin-conjugating enzyme E2M [B. mori] actin-depolymerizing factor 1 [B. mori] reptin [D. melanogaster] arginine kinase [H. armigera]

8.84/24.23

database

Mowse score(A/B); A G B indicates identity or extensive homology (p < 0.05); higher scores indicate higher confidence of identity.

to the brain. Ketone bodies further generate acetyl-CoA to supply energy for metabolic activity at pupal stage; the reaction is catalyzed by two enzymes, 3-hydroxybutyrate dehydrogenase type 2 and acetoacetyl-CoA thiolase. In this study, 3-hydroxybutyrate dehydrogenase type 2 and acetoacetyl-CoA thiolase were more abundant at the early pupal stage. Thus, the two enzymes may provide the insect with energy from specifically metabolic pathway at diapause initiation, such as biosynthesis of polyols. Another protein is ArgK, which is less abundant at diapause initiation, as indicated by 2-DE and Western blot. ArgK is a phosphotransferase that catalyzes the reaction between Larginine and ATP to produce L-phospho-arginine and ADP. It is the only phosphagen kinase in insects, and it plays a vital role as ATP-buffering systems to regulate ATP level, just like creatine kinase in vertebrate.35 Chen et al. found that ArgK was not abundant in H. armigera heads from diapausing pupae.15 In our previous work, ArgK activity was maintained at low level in diapausing pupae but rose in nondiapause pupae. This implies that high ArgK expression is associated with pupal development as a response to active metabolism and that low ArgK expression represents a slow development associated with low energy metabolism, as observed in the diapause state. Therefore, the low abundant ArgK may decrease energy metabolism at diapause initiation. 4.2. Response to Stress. Diapause insects have to confront the harsh condition of overwintering. Hsps, as molecular chaperones, protect cellular proteins from denaturation especially during stress responses. Hsps contribute to the cold tolerance of insects.36 In pupal diapause of S. crassipalpis, Hsp23 and Hsp70 are developmentally up-regulated during diapause,37,38 while Hsp90 is down-regulated at the same time.39 In the present study, Hsp21.4 and Hsp20.7 are differentially expressed at diapause initiation, and Hsp21.4 protein is more abundant in diapause brain, but Hsp20.7 is less abundant. The two proteins, which belong to a small Hsp family, may be involved in different physiological functions during diapause.40 An increase of Hsp21.4 expression may enhance cold tolerance and protect the brain, like up-regulated Hsp23 and Hsp70.36 Hsp20.7 expression decreases at the early pupal stage, as does Hsp90 expression in Helicoverpa zea;41 this result implies that Hsp20.7 may be involved in nondiapause pupal development by responding to 20-hydroxyecdysone, as reported in H. zea and D. melanogaster.42

Another stress response protein is glutathione S-transferase (GST). In the spruce budworm Choristoneura fumiferana, GST expression is higher throughout diapause and declines at the end of diapause.43 GST may protect the brain from adverse conditions. In the present study, two GST molecules were highly expressed in the diapause-destined pupal brain, suggesting that GST is closely correlated with the stress response. MnSOD is less abundant in diapause individuals but is highly expressed in nondiapause pupae. It is a member of the iron superoxide dismutase family and eliminates toxic superoxide, which is a byproduct of the electron transport chain. MnSOD may play a role in pupal development when aerobic respiration is used. The PPP is up-regulated in hypometabolism states in vertebrates; it seems to be a common mechanism to regulate developmental arrest in all eukaryotic organisms.44 As discussed above, PPP can generate NADPH to synthesize cryoprotectants, but NADPH can also be used to produce reducing forms of antioxidants. Therefore, we conclude that the PPP activity may maintain NADPH product in diapause individuals for use in antioxidant defense. In addition, bombyrin expression in diapause-destined brains is about 5-fold higher than in nondiapause-destined brains. Bombyrin belongs to lipocalin family; it is similar to lazarillo and human apolipoprotein D.45 The bombyrin homologue lazarillo is a GPI (glycosylphosphatidylinositol)-linked protein in the central nervous system of the grasshopper Schistocerca americana, and it plays a key role in the pioneer neuron development.46,47 Also, the Drosophila lazarillo is reported in the central nervous system, and overexpression of glial or neural lazarillo represses growth and promotes oxidation stress resistance.48-50 Thus, bombyrin may serve as an antioxidant that increases oxidative resistance and represses cell growth in insect diapause brains to extend the life span. 4.3. Signal Transduction. In this study, we found that a series of regulatory proteins changed at diapause initiation except energy metabolic and stress response proteins. Calmodulin, which is less abundant at diapause initiation, is an important regulatory protein that adjusts a wide range of cellular process and development.51 This protein can bind second messenger calcium to affect a number of target proteins. Another protein that is low abundant at diapause, phospholipase A2, is one of the downstream proteins of calmodulin signal.51 We found that calmodulin protein kinase II (CaMKII) was also decreased at the mRNA and protein levels Journal of Proteome Research • Vol. 9, No. 10, 2010 5059

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Lu and Xu

Figure 4. Representative PMF and MS/MS spectra. Spot 21 is identified as ArgK. (A) PMF spectrum; (B) MS/MS spectrum.

at diapause initiation (data not shown). Apparently, calmodulin-dependent signaling, which is required for development, is halted at the onset of diapause. Interestingly, three actin-related proteins were differentially expressed at diapause initiation in this study. ADF1 and actinrelated protein 66B were less abundant at diapause initiation, whereas another protein F-actin capping protein subunit alpha was more abundant. ADF1 can stimulate the depolymerization of actin filaments to promote actin turnover,52 and actin-related protein 66B, which belongs to the Arp2/3 complex, participates 5060


in forming new actin branches off existing branches.53 F-actin capping protein subunit alpha is an actin-binding protein that controls actin filament length by binding to the fast-growing actin ends and preventing filament elongation.53 Changing these three proteins at diapause initiation may depress actin mobility and arrest cell growth, and it is consistent with a cell cycle arrest of S. crassipalpis pupal brain in G0/G1.54 Previous studies on hypometabolism (e.g., anoxia and hibernation) showed that protein phosphorylation is the dominant regulatory mechanism through changing enzymes activity,

research articles

Proteome and Phosphoproteome at Diapause Initiation Table 2. Identification of Differentially Expressed Phosphoproteins spot no.

ratio

accession no.


description


Mowse scorea

EST_Har

2

95/29

EST_Har

2

137/47

NCBInr

4

149/47

EST_others

3

172/59

EST_Har

2

137/47

EST_others

3

172/59

EST_Har

2

95/29

EST_others

2

84/59

NCBInr NCBInr

4 1

149/47 74/47

database

Phosphoproteins in DP2/NP1 pa2

1.57

gi|162952033

pa1

1.52

gi|153792659

pa3

5.56

gi|148298816

pa4

2.70

gi|24641032

Proteins More Abundant in Diapause ribosome-associated 4.87/33.50 protein P40 [B. mori] actin-depolymerizing 6.17/17.23 factor 1 [B. mori] Proteins Less Abundant in Diapause proteasome 26S non-ATPase 4.76/39.50 subunit 4 [B. mori] protein phosphatase 4 4.32/66.81 regulatory subunit 2-related protein [D. melanogaster] Phosphoproteins in DP4/NP2

pb1

1.86

gi|153792659

pb7

2.61

gi|24641032

pb4

2.50

gi|162952033

pb6

1.84

gi|30421352

pb5 pb8

1.83 1.82

gi|148298816 gi|156544285

a

Proteins More Abundant in Diapause actin-depolymerizing 6.17/17.23 factor 1 [B. mori] Proteins Less Abundant in Diapause protein phosphatase 4 4.32/66.81 regulatory subunit 2-related protein [D. melanogaster] ribosome-associated 4.87/33.50 protein P40 [B. mori] Hsc-70-interacting 5.18/42.48 protein-like protein [Drosophila yakuba] proteasome 26S non-ATPase subunit 4 [B. mori] 4.76/39.50 PREDICTED: similar to 4.30/66.57 protein phosphatase 2c gamma [Nasonia vitripennis]

Mowse score(A/B); A G B indicates identity or extensive homology (p < 0.05); higher scores indicate higher confidence of identity.

ion channel, and protein synthesis rate.55 During changes in development, protein kinases and protein phosphatases play a key role in ‘turning on’ and ‘turning off’ target pathways. In this study, except the global protein phosphorylation level decreased at diapause initiation, a protein phosphatase, serine/ threonine protein phosphatase 1 beta-catalytic subunit (PP1c), was highly expressed at the start of diapause. PP1c is a major protein phosphatase that regulates multicellular functions, such as cell division, metabolism, protein synthesis, and cytoskeletal reorganization.56 PP1 activity is low in response to cold exposure in the larvae of the goldenrod gall fly, Eurosta solidaginis.57 However, we propose that PP1c in the diapause pupal brain regulates some the real-time activity of enzymes and proteins by dephosphorylation. In addition, protein phosphatase 4 regulatory subunit 2-related protein (PP4R2r, a member of the serine/threonine protein phosphatase family) does not change at the protein level but is dephosphorylated at diapause initiation. As a cell-cycle regulator, PP4R2r is involved in cell mitosis and increases cell size in D. melanogaster.58 The cell cycle arrest in H. armigera diapause-destined brain may be caused by the two phosphorylation regulators, as observed other diapause insects,54,59 although the exact mechanism still needs to be proved. Meanwhile, phosphorylated ADF1 expression increases at diapause initiation, and the phosphorylated-ADF1 blocks out its activity.60 As described above, the ADF1 gene expression is down-regulated at the early pupal stage. Thus, ADF1 activity is efficiently inhibited to reduce actin mobility through both gene expression and phosphorylation levels. In addition to protein phosphorylation, protein ubiquitination is also an important PTM mechanism that marks protein for degradation. In the brine shrimp Artemia franciscana,

ubiquitin-meditated proteolysis is blocked during hypometabolism because the level of ubiquitin-conjugate proteins in anoxic embryos drops to 7% of normoxic values.61 Two ubiquitin-conjugate enzymes, E2M and effete, are less abundant at diapause initiation. To the best of our knowledge, the global translation rate is lower in diapause individuals to save energy,6 and the total number of protein in diapause type brain is similar to that of nondiapause pupae in H. armigera, as also reported in S. crassipalpis.8 These results implicate that a strategy for insect diapause is a decrease in both protein synthesis and proteolysis. The proteolysis may be caused by certain enzymes, such as ubiquitin-conjugating enzyme E2M and effete. Therefore, we conclude that the decreased protein ubiquitination may play a key role in maintaining protein activity for diapause insect.

5. Conclusion Diapause initiation is a complex biological process rather than a simple gene expression shutdown. During this process, some upstream signal factors (e.g., neuropeptides and neurohormones) are released to regulate a cluster of diapause-related gene expression in the brain. On the basis of the evidence shown above, the mechanisms may include: (1) altering the metabolic pathways, such as energy utilization (e.g., using ketone bodies as fuel) and decreases in anabolism, and the specific synthesis of substances for protecting organs or tissues from harsh conditions during diapause, such as cryoprotectants (glycerol, sorbitol, and trehalose), chaperones, and antioxidants (Hsps, GST, and bombyrin); (2) halting the developmental signal transduction pathway through up- and down-regulating gene expression by regulatory genes and ubiquitination; and (3) regulating biochemical mechanisms for diapause initiation Journal of Proteome Research • Vol. 9, No. 10, 2010 5061

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Figure 5. Identification of differentially expressed proteins in diapause- and nondiapause-destined pupae. (A) The closed view of 11 selected proteins from the total proteome. ADF1 is found in two different spots; ADF1a is phosphorylated, and ADF1b is not. (B) RTPCR analysis. Nine selected genes that were differentially expressed at the protein level were checked at the mRNA level, and actin was used as an internal control. (C) The PCR products were quantified by an image analyzer (Molecular Imaging Software, Kodak) and normalized to actin expression. Each value represents the relative amount of mRNA expression in (DP2/NP1) and (DP4/NP2) with three repeated experiments (average ( SE). (D) Western blot analysis. Proteins were separated on a 12% SDS-PAGE gel and immunoblotted with either anti-MnSOD serum (diluted 1:3000) or anti-ArgK serum (diluted 1:2000). Actin was used as an internal control. (E) Changes of global protein tyrosine phosphorylation. Proteins were lysed and separated on a 6-12% SDS-PAGE gel, and phosphotyrosine antibody (pY) was used (diluted 1:1000). 5062



Figure 6. A schematic drawing for cryoprotectant biosynthesis in H. armigera brain. Aldolase, MADH, and pentose phosphate pathway (PPP) are indicated in bold. The cryoprotectants (glycerol and sorbitol) are shown in italics.

through protein phosphorylation. Thus, insect development will be arrested by integrating these mechanisms at the early pupal stage. However, this hypothesis needs further confirmation at the genomic and transcriptomic level to help us understand the molecular mechanism of diapause in detail.

Acknowledgment. We thank Qi Zhang for her assistance in the sample collection and MS analysis. This study was supported by a Grant-in-Aid for the Natural Scientific Foundation (30730014) from the National Natural Science Foundation of China, and the Major State Basic Research Developmental Program (2006CB102001) from the Ministry of Science and Technology of China. Supporting Information Available: Supplementary Table 1, the gene-specific primers used in RT-PCR. Supplementary Figure 1, distribution of the differentially expressed total proteins and phosphoproteins among different GO categories. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Kostal, V. Eco-physiological phases of insect diapause. J. Insect Physiol. 2006, 52 (2), 113–27. (2) Denlinger, D. L. Regulation of diapause. Annu. Rev. Entomol. 2002, 47, 93–122. (3) Denlinger, D. L.; Yocum, G. D.; Rinehart, J. P. Hormonal control of diapause. In Comprehensive Insect Molecular Science; Gilbert, L. I., Iatrou, K., Gill, S., Eds.; Elsevier: Amsterdam, 2005; Vol. 3, pp 615-50. (4) Velde, S. V.; Badisco, L.; Marchal, E.; Broeck, J. V.; Smagghe, G. Diversity in factors regulating ecdysteroidogenesis in insects. In Ecdysone: Structures and Functions; Smagghe, G., Ed.; Springer: The Netherlands, 2009; pp 283-315. (5) Denlinger, D. L. Molecular regulation of insect diapause. In Environmental Stressors and Gene Responses; Storey, K. B., Storey, J., Eds.; Elsevier Science: Amsterdam, 2000; pp 259-75. (6) Joplin, K. H.; Yocum, G. D.; Denlinger, D. L. Diapause specific proteins expressed by the brain during the pupal diapause of the flesh fly, Sarcophaga crassipalpis. J. Insect Physiol. 1990, 36 (10), 775–83. (7) Flannagan, R. D.; Tammariello, S. P.; Joplin, K. H.; Cikra-Ireland, R. A.; Yocum, G. D.; Denlinger, D. L. Diapause-specific gene expression in pupae of the flesh fly Sarcophaga crassipalpis. Proc. Natl. Acad. Sci. U.S.A. 1998, 95 (10), 5616–20. (8) Li, A. Q.; Popova-Butler, A.; Dean, D. H.; Denlinger, D. L. Proteomics of the flesh fly brain reveals an abundance of upregulated heat shock proteins during pupal diapause. J. Insect Physiol. 2007, 53 (4), 385–91.

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PR100356T

Proteomic and Phosphoproteomic Analysis at Diapause Initiation in

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