Spatial Proteomics Sheds Light on the Biology of ...

Current Proteomics, 2012, 9, 000-000

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Spatial Proteomics Sheds Light on the Biology of Nucleolar Chaperones Mohamed Kodiha, Michael Frohlich and Ursula Stochaj* Department of Physiology, McGill University, 3655 Promenade Sir William Osler, Montreal, PQ, H3G 1Y6, Canada Abstract: Within the nucleus, the nucleolus is a dynamic compartment which is critical to maintain cellular homeostasis under normal, stress and disease conditions. During the last years, proteomics research provided new information on the complexity of nucleolar proteomes. These studies also established that many chaperones, co-chaperones and other factors involved in proteostasis associate with nucleoli in the absence of stress or disease. Moreover, quantitative proteomics demonstrated that physiological and environmental changes alter the nucleolar profile of chaperones and co-chaperones. At present, the emphasis has shifted towards sophisticated in-depth analyses of the nucleolar proteome. As such, turnover and posttranslational modifications are now quantified for individual proteins that associate with nucleoli. This large body of work generated new insights into the sumoylation, phosphorylation and acetylation of the nucleolar proteome. At the same time, we have gained a better understanding of the nucleolar organization, as novel subcompartments were identified within the nucleolus that are induced by physiological and other forms of stress. Notably, some of these subcompartments are also enriched for chaperones. To review these results, we will focus on recent studies that analyzed the nucleolar proteome, and particular emphasis will be given to nucleolar chaperones. Despite remarkable progress in the field, crucial questions regarding the physiological relevance of nucleolar chaperones remain to be answered in the years ahead. We conclude our update by discussing these future directions in the context of the latest developments in the nucleolar and chaperone fields.

Keywords: Proteomics, nucleolus, chaperones. INTRODUCTION This current update of a previously published review [1] presents new information on chaperones and their co-factors in the nucleolus. In particular, we will focus on those new insights that were gained by proteomics, but complementary approaches from other fields will also be included. Here, we refer to molecular chaperones as proteins that provide a set of biological activities which are crucial to proteostasis. Thus, molecular chaperones fold or unfold polypeptides, and promote the assembly or disassembly of higher order structures. These properties make chaperones and co-chaperones key components of cell physiology that are therapeutic targets to improve human health or aging [2-11]. It is wellestablished that nucleoli, like chaperones, modulate aging and the pathophysiology of many diseases [12-16]. While the importance of chaperones for cellular proteostasis is generally accepted, much less is known about the compartmentspecific chaperone activities that contribute to these processes. This applies in particular to the biological roles that chaperones play in the nucleolus. In recent years, large datasets have become available for the nucleolar proteome, which contains several thousand different proteins, including many chaperones, co-chaperones and other factors involved in proteostasis [17-20]. Whereas earlier studies generated a comprehensive inventory of nucleolar proteins in distinct organisms, current work focuses on *Address correspondence to this author at the Department of Physiology, McGill University, 3655 Promenade Sir William Osler, Montreal, PQ, H3G 1Y6, Canada; Tel: 514-398-2949; Fax: 514-398-7452; E-mail: [email protected]

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quantitative differences that arise from pathological changes or pharmacological intervention [21-26]. Moreover, the posttranslational modifications of nucleolar proteins and their impact on nucleolar organization and function have been the subject of intense research [27-29]. Here, we will discuss these experiments as they relate to nucleolar chaperones and other protein folding factors. NUCLEOLI - MULTIFUNCTIONAL AND DYNAMIC COMPARTMENTS A comprehensive description of the nucleolus, including its dynamic organization, function, assembly and disassembly can be found in several publications [30-40]. In addition, our previous review [1] presented information on nucleolar biology, as well as a detailed list of chaperones and their cofactors that associate with nucleoli. For the current update, we will only briefly summarize the background information that is pertinent to the nucleolus and chaperones. Within the nucleus, nucleoli are organized around multiple copies of rDNA genes, thereby providing a specialized compartment for the transcription and processing of 45S prerRNA as well as the biogenesis of ribosomal subunits. Aside from these activities, nucleoli assemble signal recognition particle, regulate cell cycle progression, apoptosis and the response to stress. With respect to human health, nucleoli are implicated in viral replication [41] and linked to tumor cell biology on multiple levels. This includes the regulation of the tumor suppressor protein p53 and nucleolar hypertrophy, a characteristic feature of many cancer cells [42-45]. Mammalian nucleoli can be divided into at least three sub-compartments with distinct biological activities (Fig. 1). ©2012 Bentham Science Publishers

2 Current Proteomics, 2012, Vol. 9, No. 3

Kodiha et al.

Fig. (1). Organization of the nucleolus in subcompartments. A schematic representation depicts three nucleoli that reside within the nucleus (blue). The magnified view of one nucleolus illustrates fibrillar centers (red), dense fibrillar components (purple), the granular component (gray/yellow) and the nucleolar aggresome (black), a subcompartment induced by proteasome inhibitors.

Specifically, fibrillar centers (FC) are located within dense fibrillar components (DFC); both FC and DFC are surrounded by the granular component (GC). More recent work identified additional subcompartments of the nucleolus, the intranucleolar body and the nucleolar aggresome (Fig. 1, [38, 46, 47, 48]). While intranucleolar bodies are found predominantly in S-phase and could control pre-rRNA synthesis, nucleolar aggresomes are induced by treatment with proteasome inhibitors, such as MG132. Interestingly, the nucleolar aggresome not only contains multiple chaperones and conjugated ubiquitin, but also poly(A)-RNA [38]. Together, these studies further confirm the model of the nucleolus as a highly dynamic compartment whose functional organization is altered by physiological and environmental stress. PROTEIN FOLDING FACTORS IN THE NUCLEOLUS As described previously [1], the term “chaperone” is used by us to describe heat shock proteins, co-chaperones and other factors that promote polypeptide folding or turnover. Multitasking nucleolar proteins and RNA chaperones were discussed elsewhere and will not be part of the current update [49, 50]. Using proteomics-based techniques, A. Lamond’s group produced an extensive list of human nucleolar proteins that is available as a searchable database, NOPdb [51]. This database contains a large number of factors that are involved

in proteostasis. For example, all heat shock protein families and chaperonins are represented in nucleoli, including hsp90, hsp70, several DnaJ proteins (hsp40), hsp110, small heat shock proteins, class I and II chaperonins and other folding factors, which we listed earlier [1]. In many, but not all cases, evidence obtained by proteomics was verified by independent approaches, such as immunolocalization. Collectively, evidence from many groups suggested that nucleoli harbor a chaperone network which is regulated by changes in cell physiology. Current proteomics and other work continue to substantiate this model. Our update will focus on these recent studies and summarize the new insights into the dynamics and posttranslational modifications of nucleolar chaperones. We begin by briefly describing the folding factors that are relevant to our review; a more detailed discussion can be found elsewhere [1]. On the basis of their molecular mass and other properties, heat shock proteins (hsps) are separated into different families, which distinguish between hsp90 (HSP C), hsp70 (HSP A), hsp40 (DnaJ), small hsps (HSP B), hsp60 (HSP D) and hsp110 (HSP H) [52-55]. The synthesis of many, but not all, heat shock proteins increases when cells are challenged by stress. Hsp90s promote protein folding, trafficking and degradation. As protein kinases are among their major clients [56], hsp90s are indispensable for cell signaling. By interacting

Spatial Proteomics Sheds Light on the Biology of Nucleolar Chaperones

Current Proteomics, 2012, Vol. 9, No. 3

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Fig. (2). Hsc70 associates with nucleoli under stress and normal growth conditions. (A) NIH3T3 fibroblasts were heat-stressed for 1 h and allowed to recover for 3 h at 37°C. In fixed cells, hsc70 was located by indirect immunofluorescence. Nuclei were stained with 4',6diamidino-2-phenylindole (DAPI), and images acquired by confocal microscopy. Note that hsc70 concentrated in nucleoli when cells recovered from heat shock, but the chaperone was also detected in nucleoli of unstressed cells (arrowheads). (B) Unstressed MCF7 breast cancer cells were stained for hsc70 (red) and the nucleolar marker protein fibrillarin (green). Confocal images were used for 3D reconstruction with Imaris software. One nucleolus was enlarged to visualize hsc70 which resided in the vicinity of fibrillarin (arrowheads).

with different co-chaperones (Ahsa1/Aha1, Cdc37, HOP/Sti) and additional folding factors (large peptidyl-prolylisomerases), the actions of hsp90 are coordinated with other chaperones, in particular hsp70s. Besides folding de novo synthesized polypeptides, hsp70s also control protein degradation and trafficking [8, 57-59]. In response to stress, the abundance of many hsp70s increases; depending on the stress, this applies as well to the constitutively synthesized hsc70 (heat shock cognate protein 70) [56]. Apart from changes in abundance, stress causes a redistribution of hsp70s. For example, hsp70 family members accumulate in nucleoli when cells recover from heat shock (Fig. 2A; [60-

63]). On the other hand, hsp70s are also present in nucleoli of unstressed cells; even without stress, hsc70 resides in the vicinity of the nucleolar marker protein fibrillarin (Fig. 2B, arrowheads). In addition to Ahsa1, Cdc37 and HOP, other cochaperones associate with nucleoli. This includes hsp110 (hsp105, hspH) and several members of the DnaJ and Bag families. Moreover, small heat shock proteins, the doublering forming class I and II chaperonins [64], prefoldins [65], peptidyl prolyl cis-trans-isomerases [66], protein disulfide isomerases [67], calreticulin, calnexin, and components of


the ubiquitination, sumoylation or protein degradation pathways are located in nucleoli. IDENTIFICATION OF NUCLEOLAR TARGETING SEQUENCES IN CHAPERONES During the past two years, there has been significant progress in predicting nucleolar localization sequences (NoLSs) [68-70], and a nucleolar localization sequence detector “NoD” was developed as a web server that identifies candidate NoLSs [70]. As discussed elsewhere [50], NoD predicts two possible NoLSs for hsp90, but fails to do so for several other chaperones or co-chaperones. These observations are in accordance with the difficulties in recognizing potential NoLSs in proteins that only transiently interact with nucleoli [68]. Therefore, spatial proteomics on the nucleolus, combined with other approaches, remains critical to define the nucleolar chaperone network. For instance, a combination of cell and molecular biology methods identified the stressdependent NoLS in hsc70 [62]. SPATIAL PROTEOMICS PROVIDES COMPREHENSIVE INFORMATION ON NUCLEOLAR CHAPERONES AND THEIR TURNOVER Spatial proteomics “measures the subcellular distribution of the proteome” [71] and, in combination with stable isotope labeling with amino acids in cell culture (SILAC) [72], has been critical to the understanding of nucleolar biology ([73] and references therein). SILAC was used to determine the distribution of more than 8,000 HeLa cell proteins between cytoplasm, nucleoplasm and nucleolus and their turnover in the three different compartments [74]. Table 1 depicts data for the subcellular chaperone distribution and the localization that was assigned by Boisvert et al. [74]. Although many of the listed chaperones scored as “cytoplasmic”, they were included in our tables, because other studies demonstrated their association with nucleoli ([1, 49] and references therein). Table 1 lists the peptide ion intensities in whole cells, cytoplasm, nucleoplasm and nucleolus as they are available in the original publication [74]. (Peptide ion intensities represent the number of ions that are derived from ionized peptides of a particular protein. When combined with SILAC, peptide ion intensities can provide quantitative information on the relative abundance of a protein in different samples.) Based on the numbers published in [74], we calculated the relative abundance in nucleoli as nucleolar/whole extract (No/Whole) and nucleolar/nucleoplasm ratio (No/Nuc). A high No/Nuc ratio suggests that within the nucleus a protein is concentrated in nucleoli. We used an arbitrary cut-off for No/Nuc ratios of 0.1 (Table 1, bold numbers) to point out candidates that are abundant in nucleoli. According to this classification, heat shock proteins hsp704L, DnaJC13 and DnaJC16 are enriched in nucleoli of HeLa cells under nonstress conditions. It is noteworthy that, while not particularly enriched in nucleoli, hsp90AB1 and hspA8 (hsc70) are overall abundant proteins, with high peptide ion intensities for nucleoli (Table 1). This is consistent with the presence of hsc70 in nucleoli of unstressed cells (Fig. 2A, B). In addition to measuring the steady-state distribution of proteins, Boisvert et al. [74] used pulse-SILAC together with

Kodiha et al.

spatial proteomics to calculate protein turnover in whole cells, cytoplasm, nucleoplasm and nucleoli of HeLa cells. A 50% turnover value was defined as the time point at which half of the protein was turned over in a particular location. These studies led to the following conclusions: (a) on average, the 50% turnover time in whole cells is ~20 h, (b) abundant proteins have a longer half life, and (c) for some proteins the turnover time depends on their subcellular distribution. As an extension of the model proposed for protein complexes [74], chaperones may be more stable in nucleoli, if they have important functions in this compartment. Following this reasoning, Table 2 lists the turnover values for folding factors that satisfy at least one of two criteria. First, candidates have been shown previously to associate with nucleoli, either by proteomics or other methods. Second, folding factors have a 50% turnover time in nucleoli of 25 h, which is above the major peak of 22-23 h for nucleoli. Notably, the folding factors listed in Table 2, show a large variation in turnover, ranging from 0.61 h for the co-chaperone Hip to 47.69 h for USP29 (Ubiquitin carboxyl-terminal hydrolase 29). When sorted according to 50% turnover values (Fig. 3), ~20% of the proteins are below 4 hours, with the majority of folding factors falling between 8 and 28 hours. Assuming that proteins particularly stable in nucleoli are involved in critical nucleolar processes, we screened for folding factors with 25 h turnover values. Notably, candidate factors belong to different chaperone families: hsp90B2P, hsp90AA1, hspA1A/B, hsc70, DnaJC19, hsp27, hsp60, hsp10, TCP1, CCT2, CCT4, CCT6A and CCT7. In addition, several peptidyl-prolyl cis-trans isomerases and components of the ubiquitination and degradation pathways fit into this category. We previously speculated that nucleoli have a unique profile of chaperones and co-chaperones [1, 49]. If the compartment-specific stability is indeed an indicator of functional relevance, the proteins listed above suggest that a limited number of folding factors is crucial for nucleolar biology under nonstress conditions. These factors could present the basic building blocks that organize the nucleolar chaperone network. THE IMPACT OF POSTTRANSLATIONAL MODIFICATIONS ON NUCLEOLAR CHAPERONES The importance of posttranslational modifications for protein targeting and stability is undisputed. In recent years, the small ubiquitin-like modifier (SUMO) has come to the forefront, as it is not only linked to intracellular trafficking, but also to nucleolar functions and the stress response [27, 29, 34, 75]. As such, the levels of SUMO-modification increase when cells are exposed to stressors, as exemplified by heat, oxidants or proteasome inhibitors [29, 75, 76]. Among the four different SUMO isoforms in vertebrates [75], SUMO1-3 are best understood. SUMO2 and SUMO3 are ~97% identical, whereas the identity between SUMO1 and SUMO2/3 amounts to only 50%. Given the stress-induced changes in SUMOylation, it is not surprising that SUMO metabolism is linked to chaperone biology. However, several publications suggest more specific links between SUMOmodification, chaperones and nucleoli. For example, Mata


Table 1.


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Spatial proteomics quantified the subcellular distribution of protein folding factors. Information on the subcellular localization and peptide ion intensities in whole cells (Whole), cytoplasm (Cyt), nucleoplasm (Nuc) and nucleoli (No) was obtained from [74]. Table cells were left empty for peptide ion intensity, if no data were available in the original publication. The relative abundance in nucleoli was compared to whole extracts and the nucleoplasm by calculating the nucleolar/whole extract (No/Whole) and nucleolar/nucleoplasm ratio (No/Nuc). A nucleolar/nucleoplasm ratio > 0.1 is shown in bold.

Protein Accession

Gene

Description

Peptide Ion Intensity

Relative abundance No/

Localization

Whole

Cyt

Nuc

No

No/Whole Nuc

Hsp90 family IPI00382470

HSP90AA1

Isoform 2 of Heat shock protein Hsp90-alpha

cyto

79922

59883

11299

202

0.003

0.018

IPI00031523

HSP90AA1

Putative heat shock protein Hsp90-alpha A2

cyto

5951

4465

1301

17

0.003

0.013

IPI00555957

HSP90AA4P


cyto

600

777

181

4

0.007

0.024

IPI00555876

HSP90AA5P


nuc

132

0

689

0

IPI00455599

HSP90AB2P

Similar to Heat shock protein Hsp 0-beta

cyto

57852

64451

10726

325

0.006

0.030

IPI00555565

HSP90AB4P

Putative heat shock protein Hsp90-beta 4

cyto

4337

2643

1077

16

0.004

0.015

IPI00027230

HSP90B1

Endoplasmin

nuc

64215

23016

32360

685

0.011

0.021

IPI00414676

HSP90AB1

Heat shock protein Hsp90-beta

cyto

219169

141232

34246

2187

0.010

0.064

IPI00556538

HSP90B2P

Putative endoplasmin-like protein

nuc

2497

0

4947

78

0.031

0.016

IPI00555915

HSP90Bf

cyto

51080

32465

5678

52

0.001

0.009

IPI00555614

AC093768.1

Putative heat shock protein Hsp90-beta-3

cyto

628

344

289

13

0.021

0.047

IPI00030275

TRAP1

Heat shock protein 75 kD, mitochondrial

nuc

23397

1001

7546

1683

0.072

0.223

cyto

3653

2205

991

118

0.032

0.119


HSPA1L

IPI00295485

HSPA4L

Heat shock 70 kD protein 4L

cyto

2487

1627

272

156

0.063

0.574

IPI00828021

HSPA4L

Heat shock 70kD protein 4-like, isoform CRA_b

cyto

6329

1565

926

486

0.077

0.526

IPI00304925

HSPA1B; HSPA1A

Heat shock 70 kD protein 1

cyto

70890

41293

19841

597

0.008

0.030

IPI00911039

HSPA1B; HSPA1A

Highly similar to Heat shock 70 kD protein 1

cyto

9177

4747

2583

115

0.013

0.045

IPI00007702

HSPA2

Heat shock-related 70 kD protein 2

nuc

687

414

581

3

0.004

0.005

IPI00002966

HSPA4


cyto

16716

11411

3095

49

0.003

0.016

IPI00003362

HSPA5

HspA5 protein

nuc

48318

11895

33741

784

0.016

0.023

IPI00339269

HSPA6


cyto

136960

55717

41270

1483

0.011

0.036

IPI00003865

HSPA8

Isoform 1 of Heat shock cognate 71 kD protein

cyto

151650

72773

47818

1950

0.013

0.041

IPI00007765

HSPA9

Stress-70 protein, mitochondrial

nuc

123546

7978

32155

9135

0.074

0.284

IPI00922694

HSPA9


cyto

255

243

0

0

IPI00292499

HSPA14


cyto

1582

1207

151

42

0.026

0.278

IPI00439715

HSPA14

Heat shock 70kD protein 14, isoform CRA_d

nuc

482

0

345

30

0.061

0.086

IPI00000877

HYOU1

Hypoxia up-regulated protein 1

nuc

5698

3506

3892

141

0.025

0.036

DnaJs IPI00012535

DNAJA1

DnaJ homolog subfamily A member 1

cyto

3072

1786

794

135

0.044

0.170

IPI00032406

DNAJA2


cyto

4204

3024

2485

135

0.032

0.054

IPI00294610

DNAJA3

Isoform 1 of DnaJ homolog subfamily A member 3, mitochondrial

nuc

2899

141

1188

271

0.093

0.228

IPI00015947

DNAJB1

DnaJ homolog subfamily B member 1

cyto

1985

1962

618

207

0.104

0.335


Kodiha et al.

Table 1. Contd…. Protein Accession

Gene

Description

Peptide Ion Intensity Localization


Whole

Cyt

Nuc

No

No/Whole Nuc

IPI00003848

DNAJB4


cyto

651

851

194

0

IPI00024523

DNAJB6

Isoform A of DnaJ homolog subfamily B member 6

cyto

111

298

14

0

IPI00008454

DNAJB11


nuc

1003

150

1256

23

0.023

0.018

IPI00939657

DNAJB12


nuc

168

27

134

2

0.014

0.018

IPI00014400

DNAJB12


cyto

342

598

151

0

IPI00925154

DNAJC2

no

183

0

0

17

0.091

IPI00830108

DNAJC2

Isoform 1 of DnaJ homolog subfamily C member 2

cyto

1163

702

182

116

0.100

0.636

IPI00006713

DNAJC3

DnaJ homolog subfamily C member 3

nuc

475

92

277

164

0.344

0.589

IPI00402231

DNAJC5


cyto

92

87

60

0

IPI00329629

DNAJC7


cyto

1700

1096

313

130

0.077

0.417

IPI00003438

DNAJC8


nuc

1214

306

793

113

0.093

0.143

IPI00154975

DNAJC9


cyto

639

491

206

50

0.078

0.243

IPI00293260

DNAJC10


nuc

834

51

687

48

0.058

0.070

IPI00465290

DNAJC11


nuc

1490

182

649

164

0.110

0.253

IPI00307259

DNAJC13


cyto

1585

613

138

289

0.182

2.090

IPI00006433

DNAJC16


cyto

1539

1313

80

218

0.142

2.720

IPI00018798

DNAJC17


nuc

284

21

238

0

Similar to DnaJ C17

nuc

19

0

107

1

0.027

0.005

IPI00936332

0.001

IPI00025510

DNAJC18


cyto

65

119

51

0

IPI00304306

DNAJC19

Mitochondrial import inner membrane translocase subunit TIM14

nuc

3161

70

881

247

0.078

0.281

IPI00413366

DNAJC21


nuc

609

182

547

146

0.240

0.267

IPI00027909

DNAJC25

Isoform 1 of DnaJC25

cyto

202

814

157

24

0.119

0.152

IPI00022501

DNAJC27; iso

Isoform 1 of DnaJ C27

196

0

0

0

IPI00157375

DNAJC30


nuc

33

0

29

0

Other cochaperones IPI00013122

CDC37

Hsp90 co-chaperone Cdc37

cyto

2608

2072

605

110

0.042

0.181

IPI00030706

AHSA1

Activator of 90 kD heat shock protein ATPase homolog 1

cyto

11080

9613

1276

147

0.013

0.115

IPI00013894

HOP (STIP1, STI1)

Stress-induced-phosphoprotein 1

cyto

17150

11460

2574

174

0.010

0.068

IPI00871856

HOP (STIP1, STI1)

Stress-induced-phosphoprotein 1

cyto

18951

13803

2702

125

0.007

0.046

IPI00479946

STIP1

STIP1 protein

no

16

0

0

2

0.100

IPI00025156

STUB1; CHIP

Isoform 1 of STIP1 homology and U boxcontaining protein 1

cyto

417

317

144

50

0.119

0.347

IPI00032826

HIP (ST13)

Hsc70-interacting protein

cyto

8335

6517

1006

51

0.006

0.051



7


Gene

Description



Whole

Cyt

Nuc

No

No/Whole Nuc

IPI00300531

BAG1

Isoform 1 of Bag family molecular chaperone regulator 1

cyto

339

157

88

31

0.092

0.355

IPI00000643

BAG2

Bag family molecular chaperone regulator 2

nuc

3054

1016

2389

56

0.018

0.023

IPI00641582

BAG3


cyto

635

436

77

27

0.042

0.350

IPI00556027

BAG5


no

3113

156

101

911

0.293

9.046

IPI00939163

HSPH1

Isoform Alpha of Heat shock protein 105 kD

cyto

6100

4643

1124

54

0.009

0.048

IPI00794417

HSPH1

Heat shock 105kD/1

no

178

0

0

18

0.100

IPI00514983

HSPH1

Heat shock 105kD/110kD protein 1, isoform CRA_b

cyto

4521

4421

864

62

0.014

0.072

IPI00910755

HSPH1

Highly similar to Heat-shock protein 105 kD

cyto

0

39

0

0

IPI00100748

HSPBP1

Highly similar to Hsp70-binding protein 1

cyto

633

605

53

16

0.025

0.296

IPI00029557

GRPEL1

GrpE protein homolog 1, mitochondrial

nuc

5686

182

1420

434

0.076

0.305

Heat shock protein beta-1, Hsp27

cyto

50303

38652

3216

229

0.005 0.062

Small heat shock proteins IPI00025512

HSPB1

IPI00007264

HSPB8

Heat shock protein beta-8

cyto

126

80

0

8

IPI00098827

HSPB11

Heat shock protein beta-11

cyto

3458

2911

113

0

0.071

Class I chaperonins IPI00076042

HSPD1

Short heat shock protein 60 Hsp60s2

cyto

482

684

29

0

IPI00784154

HSPD1

60 kD heat shock protein, mitochondrial; GroEL

nuc

397477

27011

112744

28875

0.073

0.256

IPI00923547

HSPD1

60 kD chaperonin (Fragment)

nuc

1167

493

710

205

0.176

0.289

60 kD chaperonin

nuc

3432

164

4140

225

0.066

0.054

10 kD heat shock protein, mitochondrial; GroES

nuc

136054

10215

35511

8500

0.062

0.239

Similar to heat shock 10kD protein 1

nuc

34523

4267

10880

1951

0.057

0.179

14488

5816

260

0.012

0.045

IPI00880053 IPI00220362

HSPE1

IPI00938042 Class II chaperonins IPI00290566

TCP1

T-complex protein 1 subunit alpha

cyto

21813

IPI00297779

CCT2

T-complex protein 1 subunit beta

cyto

26166

17878

6529

319

0.012

0.049

IPI00553185

CCT3

T-complex protein 1 subunit gamma

cyto

29805

18980

7858

400

0.013

0.051

IPI00302927

CCT4

T-complex protein 1 subunit delta

cyto

33099

18767

8345

295

0.009

0.035

IPI00873222

CCT4


cyto

30640

16920

7906

322

0.011

0.041

IPI00010720

CCT5

T-complex protein 1 subunit epsilon

cyto

29547

19043

6903

256

0.009

0.037

IPI00027626

CCT6A

T-complex protein 1 subunit zeta

cyto

26186

17883

5913

256

0.010

0.043

IPI00220656

CCT6B

T-complex protein 1 subunit zeta-2

cyto

2207

1740

577

76

0.035

0.132

IPI00018465

CCT7

T-complex protein 1 subunit eta

cyto

25229

16205

6623

593

0.024

0.090

IPI00784090

CCT8

T-complex protein 1 subunit theta

cyto

16262

9321

4861

125

0.008

0.026

Additional factors involved in protein folding IPI00413778

FKBP1A

Peptidyl-prolyl cis-trans isomerase

cyto

6372

5304

310

81

0.013

0.263

IPI00219005

FKBP4

FK506-binding protein 4

cyto

16661

12715

2853

424

0.025

0.149

IPI00640341

FKBP8

Isoform 1 of FK506-binding protein 8

nuc

636

0

1864

40

0.063

0.021

IPI00303300

FKBP10


nuc

3969

2166

3837

93

0.023

0.024


Kodiha et al.


Gene

Description



Whole

Cyt

Nuc

No

No/Whole Nuc

IPI00007019

PPIL1

Peptidyl-prolyl cis-trans isomerase-like 1

cyto

854

398

395

70

0.082

0.177

IPI00300952

PPIL3


cyto

1291

1048

468

67

0.052

0.143

IPI00026519

PPIF

Peptidyl-prolyl cis-trans isomerase, mitochondrial

nuc

7622

371

2072

533

0.070

0.257

IPI00025252

PDIA3

Protein disulfide-isomerase A3

nuc

33862

8608

24811

466

0.014

0.019

IPI00893541

PDIA3

14 kD protein

nuc

26952

6140

18586

184

0.007

0.010

IPI00031479

PDIA5


cyto

275

1292

211

30

0.109

0.142

IPI00020599

CALR

Calreticulin

nuc

36436

10321

25800

272

0.007

0.011

IPI00020984

CANX

cDNA FLJ55574, highly similar to Calnexin

nuc

39690

5486

30029

779

0.020

0.026

ubiquitin C

no

72

0

0

7

0.096

IPI00798127 IPI00645078

UBA1

Ubiquitin-like modifier-activating enzyme 1

cyto

63120

45109

7033

228

0.004

0.032

IPI00791004

UBA5

Ubiquitin-activating enzyme 5 isoform 2

cyto

190

119

62

63

0.333

1.019

IPI00217407

UBR2

Isoform 4 of E3 ubiquitin-protein ligase UBR2

cyto

6338

3118

1462

582

0.092

0.399

IPI00011245

USP29

Ubiquitin carboxyl-terminal hydrolase 29

nuc

2090

268

863

221

0.106

0.256

IPI00001786

USP36

Isoform 2 of Ubiquitin carboxyl-terminal hydrolase 36

cyto

1705

1091

162

208

0.122

1.286

IPI00871372

HECTD1

HECT domain containing 1

nuc

3657

603

1336

937

0.256

0.702

IPI00328911

HECTD1

E3 ubiquitin-protein ligase HECTD1

no

2090

106

19

256

0.123

13.494

IPI00945379

NEDD4

Isoform 1 of E3 ubiquitin-protein ligase NEDD4

nuc

552

148

232

32

0.058

0.138

IPI00166784

NSMCE2

E3 SUMO-protein ligase NSE2

no

822

22

53

101

0.122

1.891

IPI00014310

CUL1

Cullin-1

nuc

718

427

675

84

0.117

0.124

IPI00008728

CLPX

ATP-dependent Clp protease ATP-binding subunit clpX-like, mitochondrial

nuc

2719

222

1055

244

0.090

0.232

IPI00219622

PSMA2

Proteasome subunit alpha type-2

cyto

15866

12655

2808

34

0.002

0.012

IPI00154509

PSMA8

Isoform 1 of Proteasome subunit alpha type-7like

nuc

708

0

187

70

0.099

0.374

IPI00215824

PSMB8

Isoform 2 of Proteasome subunit beta type-8

nuc

1698

807

932

151

0.089

0.162

IPI00299608

PSMD1

Isoform 1 of 26S proteasome non-ATPase regulatory subunit 1

cyto

4730

3480

1747

235

0.050

0.134

IPI00031106

PSMG3

Proteasome assembly chaperone 3

cyto

1473

1121

237

761

0.517

3.208

IPI00032140

SERPINH1

Serpin H1

nuc

32353

5451

13996

1340

0.041

0.096

IPI00797126

NACA

Nascent polypeptide-associated complex alpha subunit isoform a

cyto

34414

28779

3565

130

0.004

0.037

IPI00000051

PFDN1

Prefoldin subunit 1

cyto

1573

987

184

840

0.534

4.565

IPI00006052

PFDN2

Prefoldin subunit 2

cyto

3493

3366

433

175

0.050

0.404

IPI00015891

PFDN4

Prefoldin subunit 4

cyto

1151

918

215

0

IPI00015361

PFDN5

Prefoldin subunit 5

nuc

8429

6101

10258

39

0.005

0.004

IPI00005657

PFDN6

Prefoldin subunit 6

cyto

2393

2142

260

246

0.103

0.947

IPI00176469

CABC1

Isoform 1 of Chaperone activity of bc1 complexlike, mitochondrial

nuc

491

0

102

54

0.109

0.525


Table 2..


9

Turnover of protein folding factors that associate with nucleoli. Values for 50% turnover (in hours) were measured in whole cells (Whole), cytoplasm, nucleoplasm (Nuc) and nucleolus (No) [74]. Empty table cells indicate that data were not available in the original publication. Based on 50% turnover values, the relative turnover was calculated as the nucleolar/whole cell (No/Whole) and nucleolar/nucleoplasm (No/Nuc) turnover ratio.

Protein Accession

Gene

Description

50% Turnover [h]

50% Turnover [h]

50% Turnover [h]

50% Turnover [h]

Relative turnover

Relative turnover

Whole

Cytoplasm

Nucleoplasm

Nucleolus

No/Whole

No/Nuc

23.50

23.04

25.37

28.97

1.23

1.14


HSP90AA1

IPI00031523

HSP90AA1

Putative Hsp90-alpha A2

32.49

22.91

24.64

IPI00455599

HSP90AB2P

Similar to Hsp90-beta

23.53

23.77

25.14

IPI00555565

HSP90AB4P

Putative heat shock protein H

21.55

22.22

28.36

12.86

0.60

0.45

IPI00027230

HSP90B1

Endoplasmin

21.69

21.43

21.00

22.25

1.03

1.06

IPI00414676

HSP90AB1

Heat shock protein Hsp90beta

24.08

24.40

26.24

IPI00556538

HSP90B2P

Putative endoplasmin-like protein

IPI00030275

TRAP1


38.02

24.65

24.79

24.32

24.75

1.00

1.02


HSPA1L

15.16

10.82

11.27

3.10

0.20

0.28

IPI00828021

HSPA4L

21.84

22.60

21.59

2.12

0.10

0.10

IPI00304925

HSPA1B, HSPA1A


23.13

23.01

23.53

27.07

1.17

1.15

IPI00911039

HSPA1A

Highly similar to heat shock 70 kD protein 1

24.25

25.05

27.45

41.77

1.72

1.52

IPI00007702

HSPA2


20.61

17.52

22.77

IPI00002966

HSPA4


22.66

21.90

23.00

IPI00003362

HSPA5

HspA5 protein

20.64

19.52

20.13

23.69

1.15

1.18

IPI00339269

HSPA6


23.43

26.11

23.35

IPI00003865

HSPA8

Isoform 1 of heat shock cognate 71 kD protein

25.34

23.85

24.56

31.36

1.24

1.28

IPI00007765

HSPA9

Stress-70 protein

22.49

23.27

1.01

1.03

IPI00922694

HSPA9

IPI00292499

HSPA14

IPI00000877

8.12

0.78

0.74

23.02

21.86

24.69

24.69


11.30

24.69

26.40

HYOU1


22.00

20.32

22.04

IPI00012535

DNAJA1


10.39

10.83

11.00

IPI00032406

DNAJA2

DnaJ homolog, subfamily A member 2

18.25

18.44

18.29

IPI00294610

DNAJA3

Isoform 1 of DnaJ A3, mitochondrial

19.61

18.80

18.64

19.61

1.00

1.05

IPI00015947

DNAJB1


19.20

17.97

21.39

2.56

0.13

0.12

IPI00003848

DNAJB4


7.62

15.57

18.72

DnaJs


Kodiha et al.

Table 2. Contd…. 50% Turnover [h]

50% Turnover [h]

50% Turnover [h]

50% Turnover [h]

Relative turnover

Relative turnover

21.38

0.65

0.03

0.03

7.82

1.77

13.80

0.86

0.89

3.36

0.13

0.35

IPI00008454

DNAJB11


21.34

IPI00927297

DNAJB11

Putative uncharacterized protein DnaJ B11

4.41

IPI00939657

DNAJB12

DnaJ homolog, subfamily B member 12

16.04

IPI00014400

DNAJB12

DnaJ homolog, subfamily B, member 12

15.24

9.77

11.23

IPI00830108

DNAJC2


24.98

24.93

9.70

IPI00006713

DNAJC3


18.34

21.61

17.45

IPI00402231

DNAJC5


18.20

17.26

18.72

IPI00329629

DNAJC7


15.78

15.18

20.27

IPI00003438

DNAJC8


18.17

17.04

18.97

18.76

1.03

0.99

IPI00154975

DNAJC9


21.96

24.09

22.20

19.57

0.89

0.88

IPI00293260

DNAJC10


21.15

21.61

21.53

5.70

0.27

0.26

IPI00465290

DNAJC11


32.30

32.66

11.69

0.36

0.36

IPI00307259

DNAJC13


23.08

21.81

22.61

3.87

0.17

0.17

IPI00006433

DNAJC16


20.15

12.92

1.15

IPI00018798

DNAJC17


21.69

Similar to DnaJ C17

31.42

IPI00936332

15.57

0.09

22.45

IPI00025510

DNAJC18


11.13

IPI00304306

DNAJC19

Mitochondrial import , translocase subunit TIM14

24.45

22.75

IPI00413366

DNAJC21


17.61

21.53

IPI00027909

DNAJC25


22.31

21.18

0.00

IPI00157375

DNAJC30


14.61

14.61

0.00

11.13

24.81

27.02

1.11

4.43

0.25

1.09

Other co-chaperones IPI00013122

CDC37

Hsp90 co-chaperone Cdc37 21.62

19.92

21.98

IPI00030706

AHSA1

Activator of 90 kD heat shock protein ATPase homolog 1

21.00

20.76

20.30

IPI00013894

HOP (STIP1, STI1)

stress-inducedphosphoprotein 1

23.72

21.92

24.84

highly similar to Homo sapiens STIP1

23.08

22.49

26.19

Isoform 1 of STIP1 homology and U box-containing protein 1

17.71

17.79

10.40

Hsc70-interacting protein

19.32

18.35

10.47

IPI00871856

IPI00025156

IPI00032826

STUB1 (CHIP)

HIP (ST13)

11.00

0.52

0.54

11.73

0.51

0.45

0.61

0.03

0.06



11

Table 2. Contd….

IPI00300531

Bag1

50% Turnover [h]

50% Turnover [h]

50% Turnover [h]

50% Turnover [h]

Relative turnover

Relative turnover


18.79

21.12

12.84

20.20

1.08

1.57

IPI00000643

Bag2


23.64

21.86

24.20

13.29

0.56

0.55

IPI00641582

Bag3


15.43

15.09

14.96

15.41

1.00

1.03

IPI00556027

Bag5


18.31

16.82

10.05

0.55

0.60

IPI00514983

HSPH1

Heat shock 105kD/110kD protein 1, isoform CRA_b

9.83

9.77

11.09

IPI00939163

HSPH1

Isoform Alpha of heat shock protein 105 kD

20.88

20.19

22.81

IPI00100748

HSPBP1

Highly similar to Hsp70binding protein 1

19.69

22.40

22.45

IPI00029557

GRPEL1


21.96

21.80

21.38

22.24

1.01

1.04

37.31

1.97

1.91

26.07

44.92

1.72

1.72

30.29

31.78

0.93

1.05


HSPB1

Heat shock protein beta, Hsp27

18.98

18.51

19.49

IPI00098827

HSPB11

Heat shock protein beta11

18.15

19.49

6.81

60 kD chaperonin (Fragment)

26.07

Class I chaperonins IPI00923547 IPI00076042

HSPD1


44.92

44.92

IPI00784154

HSPD1

60 kD heat shock protein, mitochondrial

34.00

27.48

IPI00880053 IPI00220362

60 kD chaperonin HSPE1

IPI00938042

19.62


26.54

31.61

26.17

26.65

1.00

1.02

Similar to heat shock 10kD protein 1

28.59

34.16

29.43

26.11

0.91

0.89

Class II chaperonins IPI00290566

TCP1


24.13

22.76

25.29

32.65

1.35

1.29

IPI00297779

CCT2


24.92

22.69

26.62

30.10

1.21

1.13

IPI00553185

CCT3


22.77

22.10

25.04

12.08

0.53

0.48

IPI00302927

CCT4


26.56

24.01

33.62

31.90

1.20

0.95

IPI00873222

CCT4

T-complex protein 1, delta subunit

25.77

24.11

29.93

36.07

1.40

1.21

IPI00010720

CCT5


17.27

14.99

21.69

11.64

0.67

0.54

IPI00027626

CCT6A


23.39

22.90

24.55

26.57

1.14

1.08

IPI00220656

CCT6B

T-complex protein 1 subunit zeta-2

9.45

22.76

20.97


Kodiha et al.


50% Turnover [h]

50% Turnover [h]

50% Turnover [h]

Relative turnover

Relative turnover

29.72

1.19

1.19

IPI00018465

CCT7


25.05

22.72

25.02

IPI00784090

CCT8


23.74

22.38

24.83


FKBP1A

FKBP1A protein

24.53

24.19

3.69

IPI00219005

FKBP4


24.46

24.33

14.37

0.83

0.03

0.06

IPI00640341

FKBP8

Isoform 1 of FK506-binding protein 8

10.67

10.36

26.62

2.49

2.57

IPI00303300

FKBP10


22.05

21.53

2.19

0.10

0.10

IPI00007019

PPIL1


23.62

21.11

24.41

36.59

1.55

1.50

IPI00300952

PPIL3

Isoform 1 of Peptidyl-prolyl cis-trans isomerase-like 3

24.58

23.75

48.00

1.95

IPI00026519

PPIF

Peptidyl-prolyl cis-trans isomerase, mitochondrial

25.14

22.41

25.60

25.90

1.03

1.01

IPI00025252

PDIA3


21.50

19.64

20.97

20.96

0.97

1.00

IPI00893541

PDIA3

Putative uncharacterized protein PDIA3

20.51

23.22

20.38

4.77

0.23

0.23

IPI00031479

PDIA5


27.79

20.69

35.80

1.29

1.73

IPI00020599

CALR

Calreticulin

20.12

19.00

19.51

8.75

0.43

0.45

IPI00020984

CANX

Highly similar to calnexin

20.78

19.46

20.71

24.48

1.18

1.18

IPI00645078

UBA1

Ubiquitin-like modifieractivating enzyme 1

24.98

24.39

26.93

5.00

0.20

0.19

IPI00791004

UBA5

Ubiquitin-activating enzyme 5 isoform 2

20.42

24.35

37.46

1.83

IPI00217407

UBR2

Isoform 4 of E3 ubiquitinprotein ligase UBR2

18.55

15.74

18.86

28.98

1.56

1.54

IPI00011245

USP29


20.99

2.84

20.74

47.69

2.27

2.30

IPI00001786

USP36

Isoform 2 of ubiquitin carboxyl-terminal hydrolase 36

19.10

16.65

13.59

18.96

0.99

1.39

IPI00871372

HECTD1


19.07

18.91

18.62

9.79

0.51

0.53

IPI00328911

HECTD1


10.84

9.15

IPI00945379

NEDD4

Isoform 1 of E3 ubiquitinprotein ligase NEDD4

0.00

IPI00166784

NSMCE2

E3 Sumo-protein ligase NSE2

9.44

IPI00014310

CUL1

Cullin

18.02

IPI00008728

CLPX

ATP-dependent Clp protease ATP-binding subunit clpXlike, mitochondrial

IPI00219622

PSMA2

proteasome subunit alpha type-2

IPI00154509

PSMA8

Isoform 1 of proteasome subunit alpha type-7-like

44.51 18.99

9.22

0.98

0.49

18.39

18.26

27.73

1.54

1.52

15.56

15.88

6.89

6.68

0.43

0.97

24.98

24.39

25.57

28.60

1.14

1.12

32.65



13


50% Turnover [h]

50% Turnover [h]

50% Turnover [h]

Relative turnover

Relative turnover

IPI00215824

PSMB8

Isoform 2 of proteasome subunit beta type-8

22.53

20.87

25.83

33.25

1.48

1.29

IPI00299608

PSMD1


29.14

24.43

30.35

32.50

1.12

1.07

22.80

1.05

1.10

0.92

0.05

0.05

IPI00031106

PSMG3

Proteasome assembly chaperone 3

21.18

21.35

22.85

IPI00032140

SERPINH1

Serpin H1

21.70

21.36

20.74

IPI00797126

NACA

Nascent polypeptideassociated complex alpha subunit isoform a

18.52

19.72

20.40

IPI00000051

PFDN1

Prefoldin subunit 1

20.89

19.89

20.32

IPI00006052

PFDN2

Prefoldin subunit 2

17.96

18.12

19.31

IPI00015891

PFDN4

Prefoldin subunit 4

20.08

19.97

10.50

IPI00015361

PFDN5

Prefoldin subunit 5

20.48

19.91

21.07

IPI00005657

PFDN6

Prefoldin subunit 6

16.76

18.77

17.65

0.62

0.04

0.04

IPI00176469

CABC1

Isoform 1 of chaperone activity of bc1 complex-like, mitochondrial

2.11

1.09

2.80

1.32

2.57

Fig. (3). Turnover values of nucleolar chaperones. The 50% turnover time is depicted for individual nucleolar chaperones, co-chaperones and prefoldin. For simplicity, other folding factors in the nucleolus that are listed in Table 2 were not included.

fora et al. [76] investigated the SUMO1 pathway in HeLa cells by comparing SUMO1-modifications in controls (DMSO) to samples treated with MG132 (a proteasome inhibitor). With respect to chaperones, two different conclusions can be drawn from these experiments (Table 3). First, multiple chaperones in nucleoli are SUMO1-modified in control samples. Second, SUMO1-modification increased for some of these chaperones when MG132 was added; this scenario applied to several members of the hsp90 and hsp70 families (Table 3). The critical role of SUMO2/3 for the survival of heat stress became evident in knockdown experiments in the human osteosarcoma cell line U2OS [29]. Moreover, the authors identified a large number of proteins in HeLa cells, including multiple chaperones and other folding factors,

which respond to heat shock with dynamic changes in SUMO2-modifications. Whether or how the change in SUMO2-modification relates to the association with nucleoli was not determined in this study. However, spatial proteomics identified new SUMO1 and SUMO2/3 targets and further explored the contribution of SUMOylation to nucleolar biology in HeLa cells [27, 77]. These experiments suggest that the abundance of several nucleolar chaperones is altered when SUMO1 or SUMO2 is overexpressed (Suppl. Table 1). Interestingly, hsp90AA1 was not only modified by SUMO1, but also 1.4-fold as abundant when SUMO1 was overexpressed (Table 3, Suppl. Table 1). Although speculative at this point, it is conceivable that SUMOylation controls hsp90 turnover.


Table 3.

Kodiha et al.

SUMO1-modification of protein folding factors in nucleoli. HeLa cells were incubated with DMSO (control) or MG132, and SUMO1-modified proteins were purified from isolated nucleoli [76]. Quantitative proteomics identified chaperones and other folding factors that are modified with SUMO1 in control cells. For some proteins, SUMO1-modification was further increased with MG132, as indicated. All candidates listed were shown to be SUMOylated in at least two of three experiments [76].

Protein Accession numbers

SUMOylation increased with MG132

Protein name

Hsp90 family IPI00382470,IPI00784295

Hsp90AA1 heat shock protein 90kD alpha

YES

IPI00414676

Hsp90AB1 heat shock protein Hsp90-beta

YES

IPI00027230

Hsp90B1, Endoplasmin

IPI00030275

TRAP1 heat shock protein 75 kD, mitochondrial


HspA1A, HspA1B heat shock 70 kD protein 1

IPI00911039


YES

IPI00003362

HspA5, HspA5 protein

YES

IPI00003865

HspA8 Isoform 1 of heat shock cognate 71 kD protein

YES

IPI00007765

HspA9 Stress-70 protein, mitochondrial

YES


HspB1 heat shock protein beta-1, Hsp27


HspD1 60 kD heat shock protein, mitochondrial


TCP1 T-complex protein 1 subunit alpha

IPI00302927,IPI00873222,IPI00893358,IPI0092 1414

CCT4 T-complex protein 1 subunit delta


PDIA3 Protein disulfide-isomerase A3

IPI00299571,IPI00644989

PDIA6 Isoform 2 of Protein disulfide-isomerase A6 RPS27A;UBB;UBC ubiquitin and ribosomal protein S27a precursor

IPI00032140

SERPINH1 Serpin H1

IPI00023748,IPI00797126,IPI00797259,IPI0090 9970

Nascent polypeptide-associated complex subunit alpha

IPI00100160

CAND1 Isoform 1 of Cullin-associated NEDD8-dissociated protein 1

As an extension of the spatial proteomics studies in HeLa cells, Ahmad et al. [78] applied pulse SILAC to determine how the phosphorylation of serine, threonine or tyrosine residues impacts protein turnover in the cytoplasm, nucleoplasm or nucleolus (Table 4). Using these datasets, we focused on the nucleolus and calculated the phosphorylated/nonphosphorylated turnover ratio. According to this ratio, phosphorylation caused a drastic reduction in the nucleolar turnover time for several folding factors, such as DnaJB1, DnaJC11, FKBP4, UBR4, UBR5 and USP7, suggesting their phosphorylation diminished the stability in nucleoli. Given that DnaJB1, DnaJC11 and FKBP4 are abundant in nucleoli (Table 1), it will be interesting to define the upstream events that trigger their phosphorylation and the downstream consequences for nucleolar function. A simpli-

YES

fied model (Fig. 4) summarizes how the posttranslational modifications discussed above may modulate chaperone biology. In addition to SUMOylation and phosphorylation, lysine acetylation is a common modification for many chaperones and protein folding factors [79]. The large number of acetylated proteins has not been analyzed by spatial proteomics, but indirect evidence for the presence of acetylated chaperones in nucleoli may come from the analysis of SIRT7 [28]. SIRT7 belongs to the sirtuin family of protein deacetylases and is concentrated in nucleoli, where it is believed to control Pol I-dependent transcription. It is noteworthy that SIRT7 has little protein deacetylase activity ([28] and references therein). Nevertheless, Ser111, which is essential for



15

Fig. (4). Posttranslational modifications control the abundance and turnover of nucleolar chaperones. SUMOylation (SUMO), phosphorylation (P) and acetylation (Ac) have been reported for many protein folding factors that associate with nucleoli. Quantitative proteomics demonstrated that SUMO modification can alter chaperone (Chap) abundance; SUMOylation may also promote chaperone targeting to subnucleolar compartments. Phosphorylation alters the turnover of some chaperones in nucleoli, possibly by changing proteasome-dependent degradation or de novo chaperone synthesis. So far, the impact of other modifications, such as acetylation, is not well-defined for nucleolar folding factors.

deacetylase activity in other Sir2 proteins, may also be important for SIRT7 function. This is suggested by the observation that a S111A mutant of SIRT7 can decrease rDNA transcription. To better understand the biological role of SIRT7, HEK293 cells were stably transfected with EGFP, SIRT7EGFP or SIRT7(S111A)-EGFP, and protein complexes were purified with antibodies against EGFP. As wild type and mutant EGFP-SIRT7 concentrated in nucleoli [28], it is reasonable to assume that the isolated complexes were to a large extent present in the nucleolus. In Table 5 we assembled protein folding factors that interact with SIRT7 and fulfill at least one of two criteria: (a) the factor has been detected in nucleoli previously [1, 51] or (b) its 50% turnover time in nucleoli is 25h [74]. Interestingly, with the possible exception of SERPINH1, all of the candidates identified are also acetylated [79, 80]. How the associations between SIRT7 and protein folding factors impact nucleolar biology has yet to be analyzed in detail. As SIRT7 is believed to participate in the control of

rDNA transcription and chromatin organization [28], nucleolar folding factors could be part of larger networks related to these functions. This hypothesis is supported by the established interaction between the chaperonin CCT2 and the nucleolar protein NOLC1 (STRING database). Accordingly, the CCT2-NOLC1 association may represent a hub that connects nucleolar chaperones to the SIRT7-POLR1A-NOLC1 network, which was proposed by Tsai et al. [28]. THE IMPACT OF DNA DAMAGE ON NUCLEOLAR CHAPERONES DNA damage and damage-induced repair processes alter many cellular functions. In this context, the nucleolus is of particular interest, because it contains components that are critical to the damage response pathway [81]. Moreover, pathway components ATM, PARP1, Ku70 and Ku80 are known to form complexes with chaperones or co-chaperones [82]. In addition, nucleoli regulate the stress-dependent sta-


Kodiha et al.

Fig. (5). Effect of stress on the abundance and localization of nucleolar chaperones. DNA damage induced by pharmacological drugs (etoposide), UV irradiation, viral infection, p53 knockout or senescence can cause the overexpression or subcellular redistribution of molecular chaperones. Cyt, cytoplasm; Nuc, nucleus; No, nucleolus.

bilization of the tumor suppressor protein p53, which is a key player in the DNA damage response [31, 83]. Given that nucleoli are essential for the stress response, several laboratories analyzed how the nucleolar proteome is affected by DNA damage, which was caused by drug treatment, UV or ionizing radiation ([71, 81, 84], summarized in Fig. 5). In a first set of experiments, the human colon carcinoma cell line HCT116 was incubated with etoposide [71, 84], a topoisomerase II inhibitor that produces DNA double strand breaks. Spatial proteomics on mock- and drug-treated cells examined the localization of more than 2,000 proteins, and results relevant to nucleolar folding factors are listed in Table 6A. Aside from original data, the rightmost column depicts the drug-induced changes for the nucleolar/nucleoplasm distribution (No/Nuc). Values < 1 suggest that etoposide decreased the No/Nuc distribution, whereas numbers >1 represent a drug-dependent increase in the No/Nuc ratio. According to this assessment, etoposide drastically reduced the nucleolar association for 20 out of 44 folding factors, i.e. the value was 1 suggests the phosphorylated protein is more stable than its non-phosphorylated counterpart. Values < 0.5 or >1.5 are in bold to emphasize drastic changes in the nucleolar turnover of phosphorylated proteins.

Protein Identifier

Non-phosphorylated protein turnover

Phosphorylated protein turnover

[h]

[h]

Gene

Description

Cyto

Nuc

IPI00382470

HSP90AA1

Heat shock protein 90kD alpha (cytosolic), class A member 1 isoform 1

23.58

IPI00555565

HSP90AB4P


IPI00414676

HSP90AB1

IPI00030275

No

phosphorylated/ nonphosphorylated

Cyto

Nuc

No

No

25.27

23.04

25.37

28.97

23.60

26.62

22.22

28.36

12.86


24.66

26.64

24.40

26.24

TRAP1


22.87

24.57

24.79

24.32

24.75

IPI00828021

HSPA4L

Highly similar to heat shock 70 kD protein 4L

22.76

20.46

22.60

21.59

2.12

IPI00002966

HSPA4


22.29

22.94

21.90

23.00

IPI00003865

HSPA8

Isoform 1 of heat shock cognate 71 kD protein

23.56

24.31

31.65

23.85

24.56

31.36

0.99

IPI00007765

HSPA9


21.38

22.45

23.48

21.86

22.49

23.27

0.99

24.74

24.69

26.40

22.19

6.07

20.32

22.04

Hsp90 family

25.18

0.98

Hsp70 family

IPI00292499

HSPA14


IPI00000877

HYOU1


IPI00012535

DNAJA1


11.43

11.30

11.33

10.83

11.00

8.12

0.72

IPI00294610

DNAJA3

Isoform 1 of DnaJ homolog subfamily A member 3, mitochondrial

19.94

18.55

21.87

18.80

18.64

19.61

0.90

IPI00015947

DNAJB1


17.93

19.74

39.89

17.97

21.39

2.56

0.06

IPI00830108

DNAJC2


26.46

21.45

24.93

9.70

3.36

IPI00402231

DNAJC5


16.95

21.85

17.26

18.72

IPI00465290

DNAJC11


10.15

27.71

IPI00307259

DNAJC13


IPI00304306

DNAJC19

Mitochondrial import inner membrane translocase subunit TIM14

22.13

IPI00413366

DNAJC21


16.57

DnaJs

25.85

41.24

32.66

11.69

0.28

21.81

22.61

3.87

20.33

22.75

24.81

27.02

1.33

6.06

21.53

4.43

0.73

Other co-chaperones IPI00030706

AHSA1

Activator of 90 kD Heat shock protein ATPase homolog 1

21.22

21.29

IPI00025156

STUB1

HOP, Isoform 1 of STIP1 homology and U boxcontaining protein 1

18.06

21.33

IPI00032826

ST13

HIP, Hsc70-interacting protein

17.42

IPI00000643

BAG2

Bag family molecular Chaperone regulator 2

21.31

24.94

IPI00641582

BAG3

Bag family molecular Chaperone regulator 3

14.91

12.73

IPI00939163

HSPH1

Isoform Alpha of Heat shock protein 105 kD

20.13

22.55

17.38

11.55

20.76

20.30

11.00

17.79

10.40

18.35

10.47

0.61

21.86

24.20

13.29

15.09

14.96

15.41

20.19

22.81

1.15



19

Table 4. Contd….



[h]

[h]

phosphorylated/ nonphosphorylated


HSPB1


18.55

18.78

40.31

18.51

19.49

37.31

0.93

IPI00784154

HSPD1

60 kD Heat shock protein, mitochondrial

27.83

30.12

31.68

27.48

30.29

31.78

1.00

IPI00220362

HSPE1

10 kD Heat shock protein, mitochondrial

35.24

25.60

27.23

31.61

26.17

26.65

0.98

Class I chaperonins


TCP1


23.43

25.35

22.76

25.29

32.65

IPI00297779

CCT2


22.38

26.46

29.79

22.69

26.62

30.10

1.01

IPI00553185

CCT3


21.79

24.25

22.28

22.10

25.04

12.08

0.54

IPI00302927

CCT4


23.96

30.02

30.14

24.01

33.62

31.90

1.06

IPI00018465

CCT7


22.94

24.72

47.40

22.72

25.02

29.72

0.63

IPI00784090

CCT8


22.10

24.68

22.38

24.83


FKBP3


22.33

23.26

23.84

21.85

24.03

24.96

1.05

IPI00219005

FKBP4


24.15

29.95

19.59

24.33

14.37

0.83

0.04

IPI00303300

FKBP10


20.89

21.53

2.19

IPI00642862

PPIL4


20.15

18.41

20.29

IPI00025252

PDIA3


20.24

20.53

19.64

20.97

20.96

IPI00009904

PDIA4


19.98

21.11

20.22

21.08

8.72

IPI00299571

PDIA6

Isoform 2 of Protein disulfide-isomerase A6

20.37

21.35

21.18

21.61

22.57

IPI00645078

UBA1


24.21

26.52

24.39

26.93

5.00

IPI00023647

UBA6

Isoform 1 of Ubiquitin-like modifier-activating enzyme 6

23.62

42.63

23.97

32.92

IPI00746451

UBE2A

Ubiquitin-conjugating enzyme E2 A

10.89

12.11

13.12

13.53

13.35

IPI00013002

UBE2C

Ubiquitin-conjugating enzyme E2 C

4.44

IPI00604464

UBE3C

Isoform 1 of Ubiquitin-protein ligase E3C

18.49

25.95

20.13

26.97

13.35

IPI00005715

UBE4B

Isoform 1 of Ubiquitin conjugation factor E4 B

12.80

12.44

11.65

11.03

IPI00013241

UBL5

Ubiquitin-like protein 5

4.19

4.77

4.21

4.84

IPI00217407

UBR2

Isoform 4 of E3 Ubiquitin-protein ligase UBR2

14.05

17.96

15.74

18.86

28.98

IPI00746934

UBR4

Isoform 2 of E3 Ubiquitin-protein ligase UBR4

21.93

22.08

20.09

22.03

10.75

9.00

0.45

IPI00026320

UBR5

E3 Ubiquitin-protein ligase UBR5

19.74

19.73

27.37

9.53

19.77

11.00

0.40

IPI00797279

UHRF1

Ubiquitin-like with PHD and ring finger domains 1 isoform 2

8.72

6.80

5.35

4.00

6.74

7.96

1.49

IPI00844050

UQCC

Isoform 1 of Ubiquinol-cytochrome c reductase complex Chaperone CBP3 homolog

18.55

18.56

10.27

17.51

19.01

15.19

1.48

IPI00024664

USP5

Isoform Long of Ubiquitin carboxyl-terminal hydrolase 5

24.68

24.52

5.08

24.66

25.63

16.97

23.25

23.98

0.37

0.02

17.05

21.64

8.02

22.88

21.01

19.48

IPI00003965

USP7


26.20

IPI00221012

USP9X

Ubiquitin specific protease 9, X-linked isoform 3

17.37

IPI00291946

USP10


22.61

12.33

22.27

26.37

0.94

0.86

1.10

1.63

19.91

3.91


Kodiha et al.

Table 4. Contd….



[h]

[h]

IPI00000728

USP15


18.66

22.82

IPI00045496

USP28

Isoform 1 of Ubiquitin carboxyl- terminal hydrolase 28

IPI00902614

USP24


IPI00001786

USP36


IPI00871372

HECTD1


18.96

16.36

IPI00014310

CUL1

Cullin-1

19.37

17.43

IPI00419273

CUL4A

Isoform 1 of Cullin-4A

16.39

20.06

IPI00018968

NAE1

NEDD8-activating enzyme E1 regulatory subunit

24.51

28.04

IPI00025019

PSMB1

Proteasome subunit beta type-1

23.90

IPI00028004

PSMB3

Proteasome subunit beta type-3

IPI00299608

PSMD1

IPI00105598

3.89

19.03

34.71

nonphosphorylated

1.66

0.43

0.98

17.60 18.90

21.15

16.65

13.59

18.96

18.91

18.62

9.79

10.22

18.39

18.26

27.73

15.78

19.73

24.65

41.95

26.36

27.25

1.24

26.56

25.40

26.46

23.77

26.40

24.07

26.39

13.30


23.09

30.18

24.43

30.35

32.50

PSMD11

Proteasome 26S non-ATPase subunit 11 variant (Fragment)

19.55

35.49

45.75

20.65

34.35

2.26

0.05

IPI00549672

PSMD13

HSPC027

23.11

36.13

17.48

22.93

33.00

0.87

0.05

IPI00024821

PSMD14

26S proteasome non-ATPase regulatory subunit 14

22.48

33.67

23.52

17.27

9.31

IPI00644482

PSMG2

Proteasome assembly Chaperone 2

21.02

22.45

23.14

IPI00033130

SAE1

SUMO-activating enzyme subunit 1

26.16

36.50

25.45

34.13

IPI00917683

SUMO1

Putative uncharacterized protein SUMO1

30.89

15.02

9.71

30.87

IPI00015361

PFDN5

Prefoldin subunit 5

19.68

20.66

13.46

19.91

bradyzoites ([88] and references therein). The pathogenencoded TgNF3 has similarities to both the nucleolar multitasking protein nucleophosmin/B23 and fungal FK506binding proteins. However, since the C-terminal domain of FK506-binding proteins is missing in TgNF3, the protein is unlikely to function as peptidyl-prolyl isomerase [88]. TgNF3 is mostly nucleolar in the tachyzoite stage of the parasite, and overexpression of TgNF3-YFP increased profoundly the size of T. gondii nucleoli. At the same time, this overexpression enhanced parasite replication in vitro, but did not alter the invasion of host cells. While the full spectrum of TgNF3 functions is far from being understood, the protein may regulate chromatin organization and possibly ribosomal biogenesis [88]. Like T. gondii, the malaria-causing parasite Plasmodium falciparium belongs to the phylum Apicomplexa. It will therefore be interesting to examine whether pathogen-derived chaperones also regulate the nucleolar organization in other parasites that are relevant to human health. SENESCENCE ALTERS CHAPERONE CONCENTRATIONS IN NUCLEOLI Chaperones play a critical role in aging, and it is generally believed that chaperone functions decline as cells age. Alterations in the nucleolar morphology were reported in aging cells [89], and more recent studies suggest mecha-

19.10

21.00

phosphorylated/

24.55

19.26

8.48

2.71

0.03

0.87

21.07

nisms that link cellular aging to nucleolar proteins [90-93]. Using sodium butyrate-treated NIH3T3 fibroblasts as a model system, Kar et al. [94] analyzed senescence-induced changes of the nucleolar proteome. In response to sodium butyrate incubation, the 10 kD mitochondrial hspE1 protein (hsp10) increased to a 6.8 fold concentration in nucleoli, whereas the T-complex subunit zeta (CCT6A) rose to a 6.5fold level. Whether changes in the nucleolar concentration of hsp10 or CCT6A affect nucleolar morphology and/or function is currently unknown. Nevertheless, like environmental stress and viral infection, senescence can be added to the factors that modulate the composition of the nucleolar chaperone network (summarized in Fig. 5). DIVERSITY OF NUCLEOLI – IS THERE A CONSERVED SET OF NUCLEOLAR CHAPERONES? Although the major functions of nucleoli are evolutionary conserved, evidence continues to emerge that their composition may vary according to cell type, differentiation or developmental stage. Nucleostemin is a prominent example of a protein that is highly abundant in cancer and stem cell nucleoli, but scarce in other cell types [92, 95, 96]. Such variations in composition justify the comparison of nucleoli from different cell types of the same organism and across species. Indeed, proteomics research on human Jurkat T-cells identified candidate proteins that were not detected in nucleoli of


Table 5.


21

Identification of binding partners for SIRT7. Components that interact with the EGFP-tagged deacetylase SIRT7 were isolated by immunoaffinity purification [28]. Binding partners for wildtype (WT) or mutant (S111A) SIRT7 and the EGFP-tag were purified. For all proteins listed the interaction with wildtype EGFP-SIRT7 was preferred over the binding to EGFP (at least 1.5 fold enrichment). The S111A mutant of SIRT7 is possibly enzymatically inactive, but that has not been established [28]. Numbers for WT and S111A illustrate the fold enrichment relative to EGFP. The table depicts protein folding factors that have been detected in nucleoli according to NOPdb, were classified as nucleolar by Boisvert et al. [74] or have a 50% turnover time > 25h in nucleoli (see Table 2).

Gene

Description

WT

S111A

WT/ S111A

P54652

HSPA2


37.5

22.4

1.7

P38646

HSPA9


1.5

0.9

1.6

Q9Y4L1

HYOU1


6.5

3.3

2.0

DNAJA1


3.2

3.6

0.9

Accession Hsp70 family

DnaJs P31689

Class II chaperonins P78371

CCT2


2.9

2.7

1.1

P49368

CCT3


2.4

2.1

1.2

P48643

CCT5


3.0

3.2

0.9


3.4

2.7

1.2

Additional factors involved in protein folding P13667

PDIA4

P27797

CALR

Calreticulin

3.1

1.9

1.7

P27824

CANX

Calnexin

2.2

1.9

1.2

Q14157

UBAP2L

Ubiquitin-associated protein 2-like

1.9

2.2

0.8

Q9ULT8

HECTD1


11.5

7.7

1.5

O95714

HERC2

E3 ubiquitin-protein ligase HERC2

24.0

14.2

1.7

Q7Z6Z7

HUWE1

E3 ubiquitin-protein ligase HUWE1

5.0

3.0

1.6

Q99460

PSMD1

26S proteasome non-ATPase regulatory subunit 1

3.2

3.4

0.9

P50454

SERPINH1

Serpin H1

3.1

2.3

1.3

other cells [97]. We previously pointed out the variability in the nucleolar localization of chaperones and their co-factors [1]. Besides differences in experimental settings, these discrepancies may suggest cell-type, tissue- or species-specific chaperone profiles that support unique tasks of nucleoli. This concept of specialized nucleolar functions is supported by the nucleolar protein NOL-6 which controls the innate immunity of C. elegans [98]. Apart from such differences in nucleolar proteomes, it is possible that nucleoli of diverse origin share a dynamic chaperone network which regulates compartment-specific functions [49]. Consistent with this idea, many of the protein folding factors associated with the Jurkat cell nucleolus are also found in other mammalian nucleoli (Table 9). Moreover, data for Arabidopsis [18] demonstrate that nucleoli from widely divergent species contain protein folding factors, such as members of the hsp90, hsp70, DnaJ and chaperonin families, as well as peptidyl-prolyl isomerases. Based on these observations, it is conceivable that the nucleolar chaperone network is composed of conserved pillars and finetuned by the addition of network components that serve more specific functions.

WHICH SUB-COMPARTMENTS OF THE NUCLEOLUS CONTAIN CHAPERONES? Given the complexity of nucleolar subcompartments that include not only DFC, FC and GC, but also intranucleolar bodies, perinucleolar compartments and nucleolar aggresomes (Fig. 1, [38, 39, 48, 99]), it will be challenging to define their functional significance. With respect to chaperones, little is known about the sub- and perinucleolar compartments they occupy and the role they play in these locations. While hsp70 was detected in the DFC of heat-shocked Chironomus thummi polytene cells [100], data are scarce for other systems, but answers are beginning to emerge for mammalian cells. For example, the nucleolar aggresome is generated upon proteasome inhibition [38], and MG132induced nucleolar aggresomes contain ubiquitin, SUMOylated proteins and members of the hsp70 family. Since the SUMOylation of several chaperones increases after MG132 treatment (Table 3), it is tempting to speculate that chaperone targeting to the nucleolar aggresome is linked to this posttranslational modification. Interestingly, SUMO1 and


Table 6.

Kodiha et al.

Etoposide treatment alters the nucleolar association of many protein folding factors. (A) The publication by Boisvert et al. [71] provided data for the distribution of chaperones and other proteostasis-related factors in the nucleoplasm (Nuc), cytoplasm (Cyt) and nucleolus (No) of HCT111 cells. If no data were available, table cells were left empty. Changes in the nucleolar/nucleoplasmic distribution after etoposide (Eto) treatment are listed in the rightmost column. For many folding factors the ratio Eto/Mock was < 1, suggesting a drug-induced relocation between nucleoli and nucleoplasm. By contrast, ratios increased for DnaJA1, AHSA1 and PFDN5; they are shown in bold. (B) Effect of etoposide (Eto) on the nucleolar/cytoplasmic distribution in HCT111 p53 wild type (WT) and double knockout cells (p53-/-) [84]. Bold numbers emphasize the drastic effects of p53 double knockout on the nucleolar/cytoplasmic distribution of protein folding factors.

Mock

Gene

Description

No/Nuc

Etoposide

Eto/Mock

Ratio

Ratio

Ratio

Ratio

Ratio

Ratio

Nuc/Cyt

No/Cyt

No/Nuc

Nuc/Cyt

No/Cyt

No/Nuc

0.04

0.02

0.29

0.14

0.02

0.10

Ratio

Hsp90 family HSP90AA1

Heat shock protein Hsp90-alpha, Hsp86

HSP90AA2


HSP90AB4P


0.17

0.12

0.56

HSP90B1

Endoplasmin, Grp94

0.99

0.09

0.10

1.49

0.10

0.08

0.75

HSP90AB1

Heat shock protein Hsp90-beta, Hsp84

0.04

0.02

0.24

0.11

0.01

0.10

0.42

TRAP1


2.68

0.59

0.21

1.61

0.09

0.09

0.44

HSPA4L

Heat shock 70 kD protein 4-like protein, osmotic stress protein 94

0.23

0.41

1.51

0.22

0.13

0.70

0.46

HSPA1

Hsp70.1, Hsp70-1/Hsp70-2

0.18

0.03

0.15

0.30

0.02

0.08

0.49


0.22

0.12

0.37

0.39

0.03

0.13

0.34

HSPA4


0.10

0.06

0.30

0.19

0.11

0.14

0.48

HSPA5

78 kD glucose-regulated protein, GRP 78

1.19

0.08

0.07

0.99

0.07

0.07

1.07

HSPA6

Heat shock 70 kD protein 6, heat shock 70 kD protein B'

HSPA8

Heat shock cognate 71 kD protein, HSC70

0.15

0.02

0.10

0.29

0.02

0.08

0.80

HSPA9

Stress-70 protein, mitochondrial, GRP 75, Mortalin

2.88

0.73

0.25

1.76

0.10

0.05

0.20

HYOU1


2.08

0.15

0.09

2.06

0.11

0.10

1.06

DnaJA1

DnaJ homolog subfamily A member 1, Hdj2

0.20

0.09

0.29

0.32

0.36

0.92

3.19

DnaJA2

DnaJ homolog subfamily A member 2, Dnj3

0.23

0.07

0.34

0.26

DnaJB1

DnaJ homolog subfamily B member 1, Hsp40

0.20

0.12

0.40

0.18

0.02

0.47

1.17

DnaJC7

DnaJ homolog subfamily C member 7, TPR repeat protein 2

0.30

0.15

0.23

CDC37

Hsp90 co-chaperone Cdc37

0.10

0.06

0.46

0.12

0.02

0.28

0.61

AHSA1

Activator of 90 kD heat shock protein ATPase homolog 1, p38

0.10

0.09

0.19

0.14

0.06

0.62

3.30

HOP (STIP1)

Hsp70/Hsp90-organizing protein

0.10

0.08

0.50

0.36

0.07

0.19

0.37

HIP (ST13)

Hsc70-interacting protein, aging-associated protein 14a

0.15

0.07

0.35

0.26

0.06

0.10

0.28

BAG2

Bcl-2-associated athanogene 2

0.45

0.83

0.02

0.02

HSPH1

Hsp105, Hsp110

0.09

0.14

0.11

0.25

4.04

0.35

0.11

Hsp70 family

70.14

0.01

DnaJs

Other co-chaperones

0.14

0.62

0.40



23

Table 6. Contd….

Mock

No/Nuc

Etoposide

Eto/Mock

HSPBP1

Hsp70-binding protein 1

0.13

GrpEL1


2.53

0.96

0.40

2.10

0.09

0.06

0.15

HSPB1


0.04

0.02

0.21

0.17

0.03

0.20

0.97

HSPD1

60 kD heat shock protein, mitochondrial, Hsp60, GroEL

4.49

1.17

0.24

2.93

0.18

0.06

0.26

HSPE1

10 kD heat shock protein, mitochondrial, Hsp10, GroES

2.95

0.78

0.23

1.32

0.10

0.06

0.26

TCP1, CCT1


0.15

0.03

0.20

0.37

0.04

0.15

0.72

CCT2


0.14

0.03

0.26

0.32

0.03

0.12

0.47

CCT3


0.16

0.03

0.16

0.34

0.04

0.16

0.95

CCT4


0.16

0.04

0.21

0.37

0.03

0.11

0.54

CCT5


0.15

0.04

0.19

0.34

0.06

0.21

1.10

CCT6A


0.17

0.03

0.18

0.44

0.05

0.17

0.96

CCT7


0.15

0.04

0.25

0.36

0.05

0.16

0.64

CCT8


0.15

0.04

0.25

0.32

0.04

0.14

0.56

0.08

0.08

0.13

Class I chaperonins

Class II chaperonins

Additional factors involved in protein folding FKBP1A (FKBP1)

Peptidyl-prolyl cis-trans isomerase FKBP1A, Immunophilin FKBP12

0.06

0.06

FKBP4 (FKBP52)

FK506-binding protein 4, Peptidyl-prolyl cistrans isomerase

0.09

0.10

1.01

0.18

0.06

0.39

0.39

PDIA3 (Erp57)

Protein disulfide-isomerase A3, Disulfide isomerase ER-60

1.21

0.12

0.10

1.16

0.07

0.07

0.69

PDIA4 (Erp70)

Protein disulfide-isomerase A4, Protein ERp72

0.94

0.11

0.12

0.93

0.11

0.18

1.54

CALR

Calreticulin

0.79

0.07

0.08

0.88

0.05

0.05

0.71

CANX

Calnexin

1.89

0.18

0.11

2.26

0.18

0.11

0.98

UBA1 (UBE1)

Ubiquitin-activating enzyme E1

0.06

0.05

0.45

0.21

0.04

0.22

0.49

HECTD1


0.07

0.40

1.75

HUWE1

E3 ubiquitin-protein ligase HUWE1

0.19

0.16

0.79

CUL1

Cullin-1

0.57

0.19

0.28

RPS27A

UBC, UBA80, 40S ribosomal protein S27a, ubiquitin B

0.37

0.13

0.24

0.91

0.08

0.15

0.61

SERPINH1

47 kD heat shock protein, Rheumatoid arthritis-related antigen RA-A47

1.46

0.24

0.14

2.07

0.15

0.25

1.75

NACA

Nascent polypeptide-associated complex subunit alpha, alpha-NAC

0.04

0.05

0.85

0.10

0.08

0.41

0.48

PFDN2

Prefoldin subunit 2

0.08

0.10

0.95

0.08

0.02

0.09

0.10

PFDN3

Prefoldin subunit 3, Von Hippel-Lindaubinding protein 1

0.12

0.14

0.49

0.16

0.08

0.45

0.91

PFDN4

Prefoldin subunit 4

0.09

0.29

0.63

0.17

0.30

0.52

0.82

PFDN5

Prefoldin subunit 5

0.08

0.06

0.06

0.12

0.02

0.50

7.95

0.53


Kodiha et al.

Table 6B p53-/-

WT No/Cyt REFSEQ

Description

No/Cyt

No/Cyt

WT

WT Eto

p53

Relative changes p53-/- /

p53 Eto

WT Eto/ WT

WT

p53

No/Cyt

-/-

-/-

p53-/- Eto/ -/-

p53-/- Eto/ WT Eto

NP_002618


0.70

0.15

0.24

0.15

0.21

0.34

0.63

1.03

NP_004125

Stress-70 protein, mitochondrial, HspA9B, Mortalin-2

0.70

0.10

0.33

0.16

0.14

0.46

0.50

1.69

NP_006775


0.96

0.09

0.42

0.16

0.09

0.44

0.38

1.78

NP_005861


1.15

0.17

0.39

0.17

0.14

0.34

0.43

1.01

NP_065777


0.78

0.10

0.35

0.15

0.13

0.45

0.43

1.49

NP_821133

Peptidyl-prolyl cis-trans isomerase B

0.57

0.38

0.60

0.87

0.67

1.04

1.46

2.28

Table 7.

WS1 skin fibroblasts were irradiated with UVC light for the times indicated [81]. Data shown were obtained for one set of experiments. The comparison between treated and control samples revealed UV-dependent changes in the nucleolar association of protein folding factors. For data points that were not measured in the original publication, table cells are left empty. Note that the nucleolar association of many, but not all, folding factors increased with time. See also Fig. 6.

Gene

Protein

1h/control

3h/ control

6h/ control

16h/ control

Hsp90 family HSP90AB1

Hsp90-beta, Hsp90-alpha

HSP90B1

Endoplasmin, heat shock protein 90 kD beta

TRAP1


1.48 1.01

0.94

3.95 2.19

1.17

3.11 2.65

Hsp70 family HSPA5

78 kD glucose-regulated protein, GRP78, BiP

1.87

1.43

1.04

1.03

HSPA8

Heat shock cognate 71 kD protein, hsc70

0.78

0.89

0.90

1.66

HSPA9

Stress-70 protein, mitochondrial, GRP75, Mortalin

1.12

1.30

1.85

1.49


0.70

Co-chaperones GRPEL1

2.02

Small heat shock proteins HSPB1


0.83

1.11

1.23

3.44

Class I chaperonins HSPD1


1.23

1.34

1.96

1.87

HSPE1

10 kD heat shock protein, mitochondrial, CPN10

1.14

1.32

1.64

1.80

Class II chaperonins CCT2


1.07

1.70

CCT3


CCT4


CCT5


0.95

1.75

CCT6A


1.29

2.64

Additional factors involved in protein folding CALR

Calreticulin

0.90

0.95

1.81

3.68

CANX

Calnexin

1.26

0.72

1.78

2.16


Table 8.


25

Effect of coronavirus infectious bronchitis virus (IBV) on the abundance of protein folding factors in nucleoli. Vero cells were infected with IBV and the nucleolar proteome of infected cells was compared to mock-treated control samples [23]. Changes are listed for chaperones and other folding factors that associate with nucleoli.

Protein IDs

Protein Names

Ratio Infected/Mock


Heat shock protein Hsp90-alpha

1.55

IPI00414676


1.51

IPI00027230

Endoplasmin precursor, heat shock protein 90 kD beta member 1

0.83

IPI00030275

TRAP-1, heat shock protein 75 kD, mitochondrial precursor

2.13

IPI00304925

Heat shock 70 kD protein 1, Hsp70-1/Hsp70-2

0.85

IPI00003362

HspA5, Grp78, BIP

0.82

IPI00003865

HspA8, heat shock cognate 71 kD protein, hsc70

0.88

IPI00007765

Hsp90, Stress-70 protein, mitochondrial precursor, Mortalin

1.03

IPI00012535

DnaJ homolog subfamily A member 1, Hdj-2

1.22

IPI00154975


1.62

Hsp70 family

DnaJs


Hsp60, 60 kD heat shock protein, mitochondrial precursor, CPN60

0.92

IPI00220362

Hsp10, 10 kD heat shock protein, mitochondrial, CPN10

0.49

IPI00290566


0.96

IPI00297779


1.12

IPI00553185


1.12

IPI00873222

T-complex protein 1 subunit delta;

1.14

IPI00027626

T-complex protein 1 subunit zeta, CCT6A

1.11

Class II chaperonins


Peptidylprolyl isomerase domain and WD repeat-containing protein 1

1.17

IPI00646304

Peptidyl-prolyl cis-trans isomerase B precursor

0.92

IPI00010796

Protein disulfide-isomerase precursor, PDI

0.48

IPI00025252

Protein disulfide-isomerase A3 precursor

0.76

IPI00009904

Protein disulfide-isomerase A4 precursor

0.53

IPI00020599

Calreticulin precursor

0.66

IPI00020984

Highly similar to calnexin

1.25

IPI00179330

40S ribosomal protein S27a, ubiquitin B, Ubc

1.08

IPI00873526

SUMO1, SMT3 homolog 3

0.63

IPI00032140

Serpin H1 precursor, Collagen-binding protein, 47 kD heat shock protein

1.01

SUMO2/3 are abundant both in nucleolar aggresomes and intranucleolar bodies. The latter subcompartments are present in unstressed cells, where they become more numerous and larger following DNA damage [48]. Whether the formation of intranucleolar bodies and aggresomes is linked or whether they are functionally related will have to be explored in further studies. CONCLUSIONS AND FUTURE DIRECTIONS The past few years witnessed tremendous progress in our understanding of nucleolar biology. The development and improvement of quantitative methods, both in proteomics and other fields [73, 101], generated the tools to examine nucleolar organization and function in a rigorous fashion (Fig. 7). Accordingly, the combination of state-of-the-art

proteomics and imaging technologies will enable us to address the unresolved questions that are pertinent to nucleolar chaperones. To date, the work of many groups attests to the intricacy of nucleolar organization and function. Yet, there are obvious gaps in our knowledge that need to be filled. For instance, there is at present no unifying concept as to the localization of chaperones in nucleolar subcompartments. Despite the established association of hsp70s with the nucleolar aggresome, the residence in other subcompartments is less well defined. As chaperone distribution probably reflects subcompartment-specific functions that are related to diseases like cancer [99], the issue of chaperone localization is not trivial and deserves a comprehensive analysis.


Table 9.

Kodiha et al.

Comparison of protein folding factors in nucleoli of different cell types and species. The nucleolar proteome was analyzed (A) for human T-cells [97] and (B) Arabidopsis thaliana ([18] and Arabidopsis Nucleolar Database; http://bioinf.scri.sari.ac.uk/cgi-bin/atnopdb/home). Protein folding factors that associated with nucleoli are shown.

Part A Gene

Protein names

Hsp90 family HSP90AA1

Heat shock protein Hsp90-alpha

HSP90AA2


HSP90AB1

Heat shock protein beta (Fragment)

HSP90AB1

Heat shock protein Hsp90-beta (Hsp90, Hsp84)

HSP90B1

Endoplasmin, Grp94

Hsp70 family HSPA4


HSPA5, GRP78

78 kD glucose-regulated protein, Grp78, BiP

HSPA8, HSC70

Heat shock cognate 71 kD protein

HSPA9, GRP75

Stress-70 protein, mitochondrial, Grp75, Mortalin

HYOU1


DnaJs DNAJA1, HDJ2


DNAJA2


DNAJB1

DnaJ homolog subfamily B member 1 (Hsp40)

DNAJC9


Other co-chaperones HOP (STIP1)

Stress-induced-phosphoprotein 1, STI1, Hsc70/Hsp90-organizing protein

HSPH1 (HSP110, HSP105)

Heat shock protein 105 kD, Heat shock 110 kD protein

Class I chaperonins HSPD1


HSPD1 (HSP60)

60 kD heat shock protein, mitochondrial, Hsp60, CPN60

HSPE1

10 kD heat shock protein, mitochondrial, Hsp10, CPN10

Class II chaperonins TCP1, CCT1


CCT2


CCT3


CCT4


CCT5


CCT6A


CCT7


CCT8


Additional factors involved in protein folding FKBP4

FK506-binding protein 4 (EC 5.2.1.8), peptidyl-prolyl cis-trans isomerase

PPIA (CYPA)

Peptidyl-prolyl cis-trans isomerase A, Cyclophilin A

PPIAL4B

Peptidylprolyl cis-trans isomerase A-like 4B

PPIB (CYPB)

Peptidyl-prolyl cis-trans isomerase B

PDIA39 (ERP57)

Protein disulfide-isomerase A3 (EC 5.3.4.1)



27

Table 9. Contd.... Part A Gene

Protein names

PDIA6

Protein disulfide-isomerase A6 (EC 5.3.4.1)

CALR

Calreticulin

CANX

Calnexin

UBA1

Highly similar to ubiquitin-activating enzyme E1

UBA1 (UBE1)


UHRF1

E3 ubiquitin-protein ligase UHRF1 (EC 6.3.2.-)

USP7 (HAUSP)

Ubiquitin carboxyl-terminal hydrolase 7 (EC 3.1.2.15)

USP11


USP14


USP21


HERC1

Probable E3 ubiquitin-protein ligase HERC1 (EC 6.3.2.-)

HUWE1 (UREB1)

E3 ubiquitin-protein ligase HUWE1 (EC 6.3.2.-)

NEDD4L

E3 ubiquitin-protein ligase NEDD4-like (EC 6.3.2.-)

UBC9 ( UBE2I, UBCE9)

SUMO-conjugating enzyme UBC9 (EC 6.3.2.-)

SAE1 (AOS1, SUA1)

SUMO-activating enzyme subunit 1 (ubiquitin-like 1-activating enzyme E1A)

SUMO3 (SMT3A, SMT3H1)

Small ubiquitin-related modifier 3, SUMO-3, SUMO-2

Part B Locus

Arabidopsis Gene Descriptor

Human ortholog

At5g56010

Heat shock protein, putative

HSPCA

Heat shock 90kD protein 1

At5g02500

Heat shock protein hsc70-1

HSPA8

Heat shock 70kD protein 8 isoform 1, Hsc70

At3g44110

DnaJ protein AtJ3

DnaJA2

DnaJ subfamily A member 2

At3g13470

Chaperonin, putative

HSPD1

Heat shock 60kD protein 1 (chaperonin, mitochondrial)

At4g25340

Immunophilin/FKBP-type peptidyl-prolyl cis-trans isomeraserelated

FKBP1B

FK506-binding protein 1B (many similar hits to this family)

At3g49600

Ubiquitin-specific protease 26 (UBP26)

At5g37640

Polyubiquitin (UBQ9)

At1g45000

26S proteasome regulatory particle triple-A ATPase subunit4related

PSMC6

Proteasome 26S ATPase subunit 6

At5g40200

DegP protease

PRSS11

Protease, serine, 11

Human ortholog description

(hsp70-1)

As described in this update, proteomics generated new insights into the dynamic association of chaperones and other folding factors with nucleoli under normal, stress and disease conditions. How posttranslational modifications regulate nucleolar functions has been the focus of several studies. With the exciting insights gained for SUMOylation, phosphorylation and acetylation of the nucleolar proteome in general, it was possible to extract a large amount of data for nucleolar chaperones and their co-factors. Collectively, the results support the model that posttranslational modifications control the abundance, turnover and nucleolar association of protein folding factors (Fig. 7). It will now be necessary to integrate these analyses and determine how aging, stress and disease alter the modification of individual chaperones.

USP31 UBC

Ubiquitin-specific protease 31 Ubiquitin C

Moreover, future studies will have to examine how other posttranslational modifications impact the nucleolus. As such, methylation [102] and the stress-modulated O-GlcNAc modification [103] of nucleolar proteins warrant in-depth exploration. Linking these modifications to nucleolar function is one of the challenges that lie ahead. However, posttranslational modifications are only one aspect of the nucleolar proteome, because the presence of protein isoforms with potentially unique roles in nucleoli [78] will further increase the complexity of nucleolar biology. While these points apply to the nucleolus in general, there are specific questions that relate to nucleolar chaperones in particular. Many chaperones differ from the majority of nucleolar proteins, as they accumulate only transiently in the nucleoli of stressed cells. Although it is a well-established response to stress, we still


Kodiha et al.

Fig. (7). Strategies to analyze nucleolar chaperones. The combination of quantitative spatial proteomics and quantitative imaging has built the foundation to define the role of chaperones and other folding factors in nucleoli. To achieve this, different physiological states of the cell, i.e. normal growth, stress and disease conditions, were examined. For these conditions, multiple aspects of chaperone biology were investigated, leading to new insights into the abundance, distribution, turnover and posttranslational modification of protein folding factors. It should be noted that we expect the different aspects of chaperone biology to be interdependent; for example, phosphorylation may alter turnover in nucleoli. The studies conducted are significant, because they set the stage to define how chaperone functions are regulated in different subcellular compartments.

know little about the nucleolar targeting of chaperones and the signaling events that control their nucleolar residence. For example, chaperones help re-establish nucleolar morphology after heat shock [61], but the molecular mechanisms involved are poorly defined. Likewise, it is clear that the abundance of chaperones in nucleoli is regulated by viral infections, but the impact of disease and aging on the nucleolar chaperone network and the downstream consequences are not well characterized. So far, we obtained a glimpse of senescence-induced alterations by studying the nucleoli of cultured fibroblasts. The recent progress in quantitative proteomics and the availability of the “SILAC mouse” should enable us to advance to the next level and address diseaseand aging-related changes of the nucleolus and its chaperone profile in animal models [104].

drug targets may emerge that could be exploited for therapeutic intervention. By the same token, disease- or agingspecific changes in the nucleolar proteome may offer new avenues for drug development.

AHA

= Activator of hsp90 ATPase

Finally, the distinct composition of nucleoli, with possible variability according to physiological state, cell type, species, differentiation or development, cautions against the simplistic view of “the” nucleolus and “the’’ nucleolar chaperone network. At the same time, this diversity could provide us with unique opportunities for therapeutic intervention. For instance, if the nucleolar proteomes of parasites and host cells indeed differ, a new repertoire of pathogen-derived drug targets may emerge that could be

Bag

= Bcl2-associated athanogene

CCT

= Chaperonin containing TCP-1

DAPI

= 4',6-Diamidino-2-phenylindole

DFC

= Dense fibrillar component

EGFP

= Enhanced green fluorescence protein

FC

= Fibrillar center

In conclusion, spatial proteomics research has generated a large body of data on nucleolar proteins, including an array of protein folding factors. This sets the stage to unravel the mechanism that link aging, environmental stress or disease to changes in the nucleolar chaperone profile and to define the specific role of chaperones for nucleolar organization and function at the molecular level. ABBREVIATIONS


Current Proteomics, 2012, Vol. 9, No. 3 [8]

FKBP

= FK506-binding protein

GC

= Granular component

Hsp

= Heat shock protein

IBV

= Coronavirus infectious bronchitis virus

NoD

= Nucleolar localization sequence detector

NoLS

= Nucleolar localization sequence

NOPdb

= Nucleolar protein database

PDI

= Protein disulfide isomerases

PFDN

= Prefoldin

Pol I

= RNA polymerase I, transcribes rDNA

PPI

= Peptidyl-prolyl isomerases

[14]

SILAC

= Stable isotope labeling with amino acids in cell culture

[15]

SIRT7

= NAD-dependent deacetylase sirtuin-7

[16]

SUMO

= Small ubiquitin-like modifier

TCP

= T-complex protein

[9] [10]

[11] [12]

[13]

[17]

CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest.

[18]

ACKNOWLEDGEMENTS This research was supported by grants from NSERC, FQRNT and HSFC to US. MK was a recipient of a fellowship from the Heart and Stroke Foundation of Canada and a postdoctoral fellowship from McGill University. MF was supported by a studentship from NSERC. We thank S. Shrivastava for providing the data shown in Fig. 2A and Dr. K. Dejgaard, McGill University, Clinical Proteomics, for helpful discussions. SUPPLEMENTARY MATERIAL

[19] [20]

[21] [22]

[23]

Supplementary material is available on the publishers Web site along with the published article. REFERENCES [1]

[2] [3]

[4] [5]

[6]

[7]

Baski, P.; Kodiha, M. and Stochaj, U. Exploring the Nucleolar Proteome: Novel Concepts for Chaperone Trafficking and Function, Curr. Proteomics, 2011, 8(1), 59-82. Hartl, F.U.; Bracher, A. and Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis, Nature, 2011, 475(7356), 324332. Dancso, B.; Spiro, Z.; Arslan, M.A.; Nguyen, M.T.; Papp, D.; Csermely, P. and Soti, C. The heat shock connection of metabolic stress and dietary restriction, Curr. Pharmac. Biotechn., 2010, 11(2), 139-145. DeZwaan, D.C. and Freeman, B.C. HSP90 manages the ends, Trends Biochem. Sci., 2010, 35(7), 384-391. Macario, A.J.L.; Cappello, F.; Zummo, G. and Conway de Macario, E. Chaperonopathies of senescence and the scrambling of interactions between the chaperoning and the immune systems, Annals New York Acad. Sci., 2010, 1197, 85-93. Morrow, G.; Kim, H.-J.; Le Pecheur, M.; Kaul, S.C.; Wadhwa, R. and Tanguay, R.M. Protection from aging by small chaperones: A trade-off with cancer? Annals New York Acad. Sci., 2010, 1197, 6775. Sterrenberg, J.N.; Blatch, G.L. and Edkins, A.L. Human DNAJ in cancer and stem cells, Cancer Lett., 2011, 312(2), 129-142.

[24]

[25]

[26]

[27]

[28]

29

Voisine, C.; Pedersen, J.S. and Morimoto, R.I. Chaperone networks: tipping the balance in protein folding diseases, Neurobiol. Disease, 2010, 40(1), 12-20. Petersen, N.H. and Kirkegaard, T. HSP70 and lysosomal storage disorders: novel therapeutic opportunities, Biochem Soc. Trans., 2010, 38(6), 1479-1483. Xu, Q.; Metzler, B.; Jahangiri, M. and Mandal, K. Molecular chaperones and heat shock proteins in atherosclerosis, Am. J. Physiol. – Heart Circ. Physiol., 2012, 302(3), H506-H514. Ebrahimi-Fakhari, D.; Wahlster, L. and McLean, P. Protein degradation pathways in Parkinson’s disease: curse or blessing, Acta Neuropathologica, 2012, 124(2), 153-172. Salminen, A. and Kaarniranta, K. SIRT1 regulates the ribosomal DNA locus: epigenetic candles twinkle longevity in the Christmas tree, Biochem. Biophys. Res. Comm., 2009, 378(1), 6-9. Drygin, D.; Rice, W.G. and Grummt, I. The RNA polymerase I transcription machinery: an emerging target for the treatment of cancer, Ann. Rev. Pharmaco. Toxicol., 2010, 50, 131-156. Deisenroth, C. and Zhang, Y. Ribosome biogenesis surveillance: probing the ribosomal protein-Mdm2-p53 pathway, Oncogene, 2011, 29(30), 4253-4260. Guarante, L. Link between aging and the nucleolus, Genes Developm., 1997, 11(19), 2449-2455. Scaffidi, P.; Gordon, L. and Misteli, T. The Cell Nucleolus and Aging: Tantalizing Clues and Hopeful Promises, PLoS Biol., 2005, 3(11), e395. Scherl, A.; Coute, Y.; Deon, C.; Calle, A.; Kindbeiter, K.; Sanchez, J.C.; Greco, A.; Hochstrasser, D. and Diaz, J.J. Functional proteomic analysis of human nucleolus, Mol. Biol. Cell, 2002, 13(11), 4100-4109. Pendle, A.F.; Clark, G.P.; Boon, R.; Lewandowska, D.; Lam, Y.W.; Andersen, J.; Mann, M.; Lamond, A.I.; Brown, J.W.S. and Shaw, P.J. Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions, Mol. Biol. Cell, 2005, 16(1), 260-269. Couté, Y.; Burgess, J.A.; Diaz, J.J.; Chichester, C.; Lisacek, F.; Greco, A. and Sanchez, J.C. Deciphering the human nucleolar proteome, Mass Spectrom. Rev., 2006, 25(2), 215-234. Ahmad, Y.; Boisvert, F.-M.; Gregor, P.; Cobley, A. and Lamond, A.I. NOPdb: Nucleolar Proteome Database--2008 update, Nucleic Acids Res., 2009, 37(Database issue), D181-184. Lam, Y.W.; Evans, V.C.; Heesom, K.J.; Lamond, A.I. and Matthews, D.A. Proteomics analysis of the nucleolus in adenovirus-infected cells, Mol. Cell. Prot., 2010, 9(1), 117-130. Lam, Y.W.; Lamond, A.I.; Mann, M. and Andersen, J.S. Analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins, Curr. Biol., 2007, 17(9), 749-760. Emmott, E.; Rodgers, M.A.; Macdonald, A.; McCrory, S.; Ajuh, P. and Hiscox, J.A. Quantitative proteomics using stable isotope labeling with amino acids in cell culture reveals changes in the cytoplasmic, nuclear, and nucleolar proteomes in Vero cells infected with the coronavirus infectious bronchitis virus, Mol. Cell. Prot., 2010, 9(9), 1920-1936. Emmott, E.; Smith, C.; Emmett, S.R.; Dove, B.K. and Hiscox, J.A. Elucidation of the avian nucleolar proteome by quantitative proteomics using SILAC and changes in cells infected with the coronavirus infectious bronchitis virus, Proteomics, 2010, 10(19), 3558-3562. Emmott, E.; Wise, H.; Loucaides, E.M.; Matthews, D.A.; Digard, P. and Hiscox, J.A. Quantitative proteomics using SILAC coupled to LC-MS/MS reveals changes in the nucleolar proteome in influenza A virus-infected cells, J. Proteome Res., 2010, 9(10), 5335-5345. Munday, D.C.; Emmott, E.; Surtees, R.; Lardeau, C.H.; Wu, W.; Duprex, W.P.; Dove, B.K.; Barr, J.N. and Hiscox, J.A. Quantitative proteomic analysis of A549 cells infected with human respiratory syncytial virus, Mol. Cell. Prot., 2010, 9(11), 2438-2459. Westman, B.J.; Verheggen, C.; Hutten, S.; Lam, Y.W.; Bertrand, E. and Lamond, A.I. A proteomic screen for nucleolar SUMO targets shows SUMOylation modulates the function of Nop5/Nop58, Mol. Cell, 2010, 39(4), 618-631. Tsai, Y.-C.; Greco, T.M.; Boonmee, A.; Miteva, Y. and Cristea, I.M. Functional Proteomics Establishes the Interaction of SIRT7 with Chromatin Remodeling Complexes and Expands Its Role in Regulation of RNA Polymerase I Transcription, Mol. Cell. Prot., 2012, 11(2), 1-17.

30 Current Proteomics, 2012, Vol. 9, No. 3 [29]

[30]

[31] [32]

[33] [34] [35] [36] [37]

[38]

[39] [40]

[41] [42]

[43]

[44]

[45]

[46] [47]

[48] [49]

[50] [51] [52]

Golebiowski, F.; Matic, I.; Tatham, M.H.; Cole, C.; Yin, Y.; Nakamura, A.; Cox, J.; Barton, G.J.; Mann, M. and Hay, R.T. System-Wide Changes to SUMO Modifications in Response to Heat Shock, Sci. Signal., 2009, 2(72), ra24-. Boisvert, F.-M.; van Koningsbruggen, S.; Navascues, J. and Lamond, A.I. The multifunctional nucleolus, Nature Rev. Mol. Cell Biol., 2007, 8(7), 574-585. Boulon, S.; Westman, B.J.; Hutten, S.; Boisvert, F.-M. and Lamond, A.I. The nucleolus under stress, Mol. Cell, 2010, 40(2), 216-227. Cisterna, B. and Biggiogera, M. Ribosome biogenesis: from structure to dynamics, Intern. Rev. Cell Mol. Biol., 2010, 284, 67111. Emmott, E. and Hiscox, J.A. Nucleolar targeting: the hub of the matter, EMBO Rep., 2009, 10(3), 231-238. Finkbeiner, E.; Haindl, M.; Raman, N. and Muller, S. SUMO routes ribosome maturation, Nucleus, 2011, 2(6), 527-532. Hernandez-Verdun, D. Assembly and disassembly of the nucleolus during the cell cycle, Nucleus, 2011, 2(3), 189-194. Hernandez-Verdun, D.; Roussel, P.; Thiry, M.; Sirri, V. and Lafontaine, D.L.J. The nucleolus: structure/function relationship in RNA metabolism, Wiley Interdisciplin. Rev., 2010, 1(3), 415-431. Hiscox, J.A.; Whitehouse, A. and Matthews, D.A. Nucleolar proteomics and viral infection, Proteomics, 2010, 10(22), 40774086. Latonen, L. Nucleolar aggresomes as counterparts of cytoplasmic aggresomes in proteotoxic stress. Proteasome inhibitors induce nuclear ribonucleoprotein inclusions that accumulate several key factors of neurodegenerative diseases and cancer, Bioessays, 2011, 33(5), 386-395. Pederson, T. The nucleolus, Cold Spring Harb. Perspect. Biol, 2011, 3:a000738. Suzuki, A.; Kogo, R.; Kawahara, K.; Sasaki, M.; Nishio, M.; Maehama, T.; Sasaki, T.; Mimori, K. and Mori, M. A new PICTure of nucleolar stress, Cancer Sci., 2012, 103(4), 632-637. Hiscox, J.A. RNA viruses: hijacking the dynamic nucleolus, Nature Rev. Microbiol., 2007, 5(2), 119-127. Krastev, D.B.; Slabicki, M.; Paszkowski-Rogacz, M.; Hubner, N.C.; Junqueira, M.; Shevchenko, A.; Mann, M.; Neugebauer, K.M. and Buchholz, F. A systematic RNAi synthetic interaction screen reveals a link between p53 and snoRNP assembly, Nature Cell Biol., 2011, 13(7), 809-818. Kumazawa, T.; Nishimura, K.; Kuroda, T.; Ono, W.; Yamaguchi, C.; Katagiri, N.; Tsuchiya, M.; Masumoto, H.; Nakajima, Y.; Murayama, A.; Kimura, K. and Yanagisawa, J. Novel nucleolar pathway connecting intracellular energy status with p53 activation, J. Biol. Chem., 2011, 286(23), 20861-20869. Tsuchiya, M.; Katagiri, N.; Kuroda, T.; Kishimoto, H.; Nishimura, K.; Kumazawa, T.; Iwasaki, N.; Kimura, K. and Yanagisawa, J. Critical role of the nucleolus in activation of the p53-dependent postmitotic checkpoint, Biochem. Biophys. Res. Comm., 2011, 407(2), 378-382. Donati, G.; Bertoni, S.; Brighenti, E.; Vici, M.; Trere, D.; Volarevic, S.; Montanaro, L. and Derenzini, M. The balance between rRNA and ribosomal protein synthesis up- and downregulates the tumour suppressor p53 in mammalian cells, Oncogene, 2011, 30(29), 3274-3288. Praefcke, G.J.K.; Hofmann, K. and Dohmen, R.J. SUMO playing tag with ubiquitin, Trends Biochem. Sci., 2012, 37(1), 23-31. Latonen, L.; Moore, H.M.; Bai, B.; Jaamaa, S. and Laiho, M. Proteasome inhibitors induce nucleolar aggregation of proteasome target proteins and polyadenylated RNA by altering ubiquitin availability, Oncogene, 2011, 30(7), 790-805. Hutten, S.; Prescott, A.; James, J.; Riesenberg, S.; Boulon, S.; Lam, Y.W. and Lamond, A.I. An intranucleolar body associated with rDNA, Chromosoma, 2011, 120(5), 481-499. Baski, P.; Kodiha, M. and Stochaj, U. Chaperones and multitasking proteins in the nucleolus: networking together for survival? Trends Biochem. Sci., 2010, 35(7), 361-367. Kodiha, M. and Stochaj, U. (2012) Multitasking nucleolar proteins and chaperones, in Proteins of the Nucleolus (O'Day, D. H., and Catalano, A., Eds.), Springer, UK. NOPdb v 3.0. (accessed 2012) Daugaard, M.; Rohde, M. and Jaattela, M. The heat shock protein 70 family: Highly homologous proteins with overlapping and distinct functions, FEBS Lett., 2007, 581(19), 3702-3710.

Kodiha et al. [53]

[54]

[55]

[56]

[57] [58]

[59] [60]

[61] [62]

[63] [64]

[65] [66]

[67]

[68] [69]

[70]

[71]

[72] [73]

[74]

Vos, M.J.; Hageman, J.; Carra, S. and Kampinga, H.H. Structural and functional diversities between members of the human HSPB, HSPH, HSPA, and DNAJ chaperone families, Biochem., 2008, 47(27), 7001-7011. Kampinga, H.H. and Craig, E.A. The HSP70 chaperone machinery: J proteins as drivers of functional specificity, Nature Rev. Mol. Cell Biol., 2010, 11(8), 579-592. Kampinga, H.H.; Hageman, J.; Vos, M.J.; Kubota, H.; Tanguay, R.M.; Bruford, E.A.; Cheetham, M.E.; Chen, B. and Hightower, L.E. Guidelines for the nomenclature of the human heat shock proteins, Cell Stress Chapererones, 2009, 14(1), 105-111. Sharma, K.; Vabulas, R.M.; Macek, B.; Pinkert, S.; Cox, J.; Mann, M. and Hartl, F.U. Quantitative proteomics reveals that Hsp90 inhibition preferentially targets kinases and the DNA damage response, Mol. Cell. Prot., 2012, 11(3), M111.014654. Gidalevitz, T.; Prahlad, V. and Morimoto, R.I. The stress of protein misfolding: from single cells to multicellular organisms, Cold Spring Harb. Perspect. Biol., 2011, 3(6), sa009704. Powers, E.T.; Morimoto, R.I.; Dillin, A.; Kelly, J.W. and Balch, W.E. Biological and chemical approaches to diseases of proteostasis deficiency, Ann. Rev. Biochem., 2009, 78, 959-991. Balch, W.E.; Morimoto, R.I.; Dillin, A. and Kelly, J.W. Adapting proteostasis for disease intervention, Science, 2008, 319(5865), 916-919. Pelham, H.; Lewis, M. and Lindquist, S. Expression of a Drosophila heat shock protein in mammalian cells: transient association with nucleoli after heat shock, Philos. Trans. R. Soc. Lond. B Biol. Sci., 1984, 307(1132), 301-307. Pelham, H.R. Hsp70 accelerates the recovery of nucleolar morphology after heat shock, EMBO J., 1984, 3(13), 3095-3100. Baski, P.; Mahboubi, H.; Kodiha, M.; Shrivastava, S.; Kanagaratham, C. and Stochaj, U. Nucleolar Targeting of the Chaperone Hsc70 Is Regulated by Stress, Cell Signaling, and a Composite Targeting Signal Which Is Controlled by Autoinhibition, J. Biol. Chem., 2010, 285(28), 21858-21867. Kodiha, M.; Chu, A.; Lazrak, O. and Stochaj, U. Stress inhibits nucleocytoplasmic shuttling of heat shock protein hsc70, Am. J. Physiol. Cell Physiol., 2005, 289(4), C1034-1041. Yebenes, H.; Mesa, P.; Munoz, I.G.; Montoya, G. and Valpuesta, J.M. Chaperonins: two rings for folding, Trends Biochem. Sci., 2011, 36(8), 424-432. Lundin, V.F.; Leroux, M.R. and Stirling, P.C. Quality control of cytoskeletal proteins and human disease, Trends Biochem. Sci,. 2010, 35(5), 288-297. Pianta, A.; Puppin, C.; Passon, N.; Franzoni, A.; Romanello, M.; Tell, G.; Di Loreto, C.; Bulotta, S.; Russo, D. and Damante, G. Nucleophosmin delocalization in thyroid tumour cells, Endocrine Pathol., 22(1), 18-23. Sheaffer, K.L.; Updike, D.L. and Mango, S.E. The Target of Rapamycin pathway antagonizes pha-4/FoxA to control development and aging, Curr. Biol., 2008, 18(18), 1355-1364. Scott, M.S.; Boisvert, F.-M.; Lamond, A.I. and Barton, G.J. PNAC: a protein nucleolar association classifier, BMC Genomics, 2011, 12, 74. Scott, M.S.; Boisvert, F.-M.; McDowall, M.D.; Lamond, A.I. and Barton, G.J. Characterization and prediction of protein nucleolar localization sequences, Nucleic Acid Res., 2010, 38(21), 73887399. Scott, M.S.; Troshin, P.V. and Barton, G.J. NoD: a Nucleolar localization sequence detector for eukaryotic and viral proteins, BMC Bioinformatics, 2011, 12, 317. Boisvert, F.M.; Lam, Y.W.; Lamont, D. and Lamond, A.I. A quantitative proteomics analysis of subcellular proteome localization and changes induced by DNA damage, Mol. Cell. Prot., 2010, 9(3), 457-470. Mann, M. Functional and quantitative proteomics using SILAC, Nature Rev. Mol. Cell Biol., 2006, 7(12), 952-958. Lamond, A.I.; Uhlen, M.; Horning, S.; Makarov, A.; Robinson, C.V.; Serrano, L.; Hartl, F.U.; Baumeister, W.; Werenskiold, A.K.; Andersen, J.S.; Vorm, O.; Linial, M.; Aebersold, R. and Mann, M. Advancing Cell Biology Through Proteomics in Space and Time (PROSPECTS), Mol. Cell. Prot., 2012, 11(3), 1-12. Boisvert, F.-M.; Ahmad, Y.; Gierlinski, M.; Charriere, F.; Lamont, D.; Scott, M.; Barton, G. and Lamond, A.I. A Quantitative Spatial Proteomics Analysis of Proteome Turnover in Human Cells, Mol. Cell. Prot., 2012, 11(3), DOI 10.1074/mcp.M111.011429

Spatial Proteomics Sheds Light on the Biology of Nucleolar Chaperones [75]

[76] [77]

[78]

[79]

[80] [81]

[82]

[83] [84]

[85] [86]

[87]

[88]

[89]

Gareau, J.R. and Lima, C.D. The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition, Nature Rev. Mol. Cell Biol., 2010, 11(12), 861-871. Matafora, V.; D'Amato, A.; Mori, S.; Blasi, F. and Bachi, A. Proteomics analysis of nucleolar SUMO-1 target proteins upon proteasome inhibition, Mol. Cell. Prot., 2009, 8(10), 2243-2255. Westman, B.J. and Lamond, A.I. A role for SUMOylation in snoRNP biogenesis revealed by quantitative proteomics, Nucleus, 2011, 2(1), 30-37. Ahmad, Y.; Boisvert, F.-M.; Lundberg, E.; Uhlen, M. and Lamond, A.I. Systematic Analysis of Protein Pools, Isoforms, and Modifications Affecting Turnover and Subcellular Localization, Mol. Cell. Prot., 2012, 11(3), DOI 10.1074/mcp.M111.011429. Choudhary, C.; Kumar, C.; Gnad, F.; Nielsen, M.L.; Rehman, M.; Walther, T.C.; Olsen, J.V. and Mann, M. Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions, Science, 2009, 325(5942), 834-840. PhosphositePlus. Cell Signaling; accessed July 2012. Moore, H.M.; Bai, B.; Boisvert, F.-M.; Latonen, L.; Rantanen, V.; Simpson, J.C.; Pepperkok, R.; Lamond, A.I. and Laiho, M. Quantitative Proteomics and Dynamic Imaging of the Nucleolus Reveal Distinct Responses to UV and Ionizing Radiation, Mol. Cell. Prot., 2011, 10(10), 1-15. Keshava Prasad, T.S.; Goel, R.; Kandasamy, K.; Keerthikumar, S.; Kumar, S.; Mathivanan, S.; Telikicherla, D.; Raju, R.; Shafreen, B.; Venugopal, A.; Balakrishnan, L.; Marimuthu, A.; Banerjee, S.; Somanathan, D.S.; Sebastian, A.; Rani, S.; Ray, S.; Harrys Kishore, C.J.; Kanth, S.; Ahmed, M.; Kashyap, M.K.; Mohmood, R.; Ramachandra, Y.L.; Krishna, V.; Rahiman, B.A.; Mohan, S.; Ranganathan, P.; Ramabadran, S.; Chaerkady, R. and Pandey, A. Human Protein Reference Database 2009 update, Nucleic Acids Res., 2009, 37(suppl 1), D767-D772. Lavin, M.F. and Gueven, N. The complexity of p53 stabilization and activation, Cell Death Differ., 2006, 13(6), 941-950. Boisvert, F.-M. and Lamond, A.I. p53-Dependent subcellular proteome localization following DNA damage, Proteomics, 2010, 10(22), 4087-4097. Shaw, P. and Brown, J. Nucleoli: Composition, Function, and Dynamics, Plant Physiol., 2012, 158(1), 44-51. Taliansky, M.E.; Brown, J.W.; Rajamaki, M.L.; Valkonen, J.P.; Kalinina, N.O.; Brown, J.W.S. and Valkonen, J.P.T. Involvement of the plant nucleolus in virus and viroid infections: parallels with animal pathosystems, Advances in Virus Research, 2010, 77, 119158. Nagy, P.D.; Wang, R.Y.; Pogany, J.; Hafren, A. and Makinen, K. Emerging picture of host chaperone and cyclophilin roles in RNA virus replication, Virology, 2011, 411(2), 374-382. Olguin-Lamas, A.; Madec, E.; Hovasse, A.; Werkmeister, E.; Callebaut, I.; Slomianny, C.; Delhaye, S.; Mouveaux, T.; Schaeffer-Reiss, C.; Van Dorsselaer, A. and Tomavo, S. A novel Toxoplasma gondii nuclear factor TgNF3 is a dynamic chromatinassociated component, modulator of nucleolar architecture and parasite virulence, PLoS Pathogens, 2011, 7(3), e1001328. Adamstone, F.B. and Taylor, A.B. Nucleolar reorganization in cells of the kidney of the rat and its relation to aging, J. Morphol., 1977, 154(3), 459-477.

Received: 00 00, 2012

Current Proteomics, 2012, Vol. 9, No. 3 [90]

[91]

[92]

[93]

[94] [95]

[96] [97] [98]

[99] [100]

[101] [102]

[103]

[104]

Revised: 00 00, 2012

31

Larson, K.; Yan, S.-J.; Tsurumi, A.; Liu, J.; Zhou, J.; Gaur, K.; Guo, D.; Eickbush, T.H. and Li, W.X. Heterochromatin formation promotes longevity and represses ribosomal RNA synthesis, PLoS Genetics, 2012, 8(1), e1002473. Huang, M.; Whang, P.; Lewicki, P. and Mitchell, B.S. Cyclopentenyl cytosine induces senescence in breast cancer cells through the nucleolar stress response and activation of p53, Mol. Pharmacol., 2011, 80(1), 40-48. Oktar, P.A.; Yildirim, S.; Balci, D. and Can, A. Continual expression throughout the cell cycle and downregulation upon adipogenic differentiation makes nucleostemin a vital human MSC proliferation marker, Stem Cell Reviews, 2011, 7(2), 413-424. Vazquez-Martin, A.; Cufi, S.; Oliveras-Ferraros, C. and Menendez, J.A. Raptor, a positive regulatory subunit of mTOR complex 1, is a novel phosphoprotein of the rDNA transcription machinery in nucleoli and chromosomal nucleolus organizer regions (NORs), Cell Cycle, 2011, 10(18), 3140-3152. Kar, B.; Liu, B.; Zhou, Z. and Lam, Y. Quantitative nucleolar proteomics reveals nuclear re-organization during stress- induced senescence in mouse fibroblast, BMC Cell Biol., 2011, 12(1), 33. Meng, L.; Lin, T. and Tsai, R.Y.L. Nucleoplasmic mobilization of nucleostemin stabilizes MDM2 and promotes G2-M progression and cell survival, J. Cell Sci., 2008, 121(Pt 24), 4037-4046. Ma, H. and Pederson, T. Nucleostemin: a multiplex regulator of cell-cycle progression, Trends Cell Biol., 2008, 18(12), 575-579. Jarboui, M.A.; Wynne, K.; Elia, G.; Hall, W.W. and Gautier, V.W. Proteomic profiling of the human T-cell nucleolus, Mol. Immunol., 2011, 49(3), 441-452. Fuhrman, L.E.; Goel, A.K.; Smith, J.; Shianna, K.V. and Aballay, A. Nucleolar proteins suppress Caenorhabditis elegans innate immunity by inhibiting p53/CEP-1, PLoS Genetics, 2009, 5(9), e1000657. Pollock, C. and Huang, S. The perinucleolar compartment, Cold Spring Harb. Perspect. Biol., 2010, 2(2), a000679. Morcillo, G.; Gorab, E.; Tanguay, R.M. and Diez, J.L. Specific intranucleolar distribution of Hsp70 during heat shock in polytene cells, Exp. Cell Res., 1997, 236(2), 361-370. Kodiha, M.; Baski, P. and Stochaj, U. Computer-based fluorescence quantification: a novel approach to study nucleolar biology, BMC Cell Biol., 2011, 12, 25. Erce, M.A.; Pang, C.N.I.; Hart-Smith, G. and Wilkins, M.R. The methylproteome and the intracellular methylation network, Proteomics, 2012, 12(4-5), 564-586. Alfaro, J.F.; Gong, C.-X.; Monroe, M.E.; Aldrich, J.T.; Clauss, T.R.W.; Purvine, S.O.; Wang, Z.; Camp, D.G.; Shabanowitz, J.; Stanley, P.; Hart, G.W.; Hunt, D.F.; Yang, F. and Smith, R.D. Tandem mass spectrometry identifies many mouse brain OGlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets, Proc. Nat. Acad. Sci. USA, 2012, 109(19), 7280-7285. Zanivan, S.; Krueger, M. and Mann, M. In vivo quantitative proteomics: the SILAC mouse, Meth. Mol. Biol., 2012, 757, 435450.

Accepted: 00 00, 2012