Current Proteomics, 2012, 9, 000-000
1
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]
1570-1646/12 $58.00+.00
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
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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
4 Current Proteomics, 2012, Vol. 9, No. 3
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
Spatial Proteomics Sheds Light on the Biology of Nucleolar Chaperones
Table 1.
Current Proteomics, 2012, Vol. 9, No. 3
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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
Putative heat shock protein Hsp90-alpha A4
cyto
600
777
181
4
0.007
0.024
IPI00555876
HSP90AA5P
Putative heat shock protein Hsp90-alpha A5
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
Hsp70 family IPI00643152
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
Heat shock 70 kD protein 4
cyto
16716
11411
3095
49
0.003
0.016
IPI00003362
HSPA5
HspA5 protein
nuc
48318
11895
33741
784
0.016
0.023
IPI00339269
HSPA6
Heat shock 70 kD protein 6
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
Stress-70 protein, mitochondrial
cyto
255
243
0
0
IPI00292499
HSPA14
Heat shock 70 kD protein 14
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
DnaJ homolog subfamily A member 2
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
6 Current Proteomics, 2012, Vol. 9, No. 3
Kodiha et al.
Table 1. Contd…. Protein Accession
Gene
Description
Peptide Ion Intensity Localization
Relative abundance No/
Whole
Cyt
Nuc
No
No/Whole Nuc
IPI00003848
DNAJB4
DnaJ homolog subfamily B member 4
cyto
651
851
194
0
IPI00024523
DNAJB6
Isoform A of DnaJ homolog subfamily B member 6
cyto
111
298
14
0
IPI00008454
DNAJB11
DnaJ homolog subfamily B member 11
nuc
1003
150
1256
23
0.023
0.018
IPI00939657
DNAJB12
DnaJ homolog subfamily B member 12
nuc
168
27
134
2
0.014
0.018
IPI00014400
DNAJB12
DnaJ homolog subfamily B member 12
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
Isoform 1 of DnaJ homolog subfamily C member 5
cyto
92
87
60
0
IPI00329629
DNAJC7
DnaJ homolog subfamily C member 7
cyto
1700
1096
313
130
0.077
0.417
IPI00003438
DNAJC8
DnaJ homolog subfamily C member 8
nuc
1214
306
793
113
0.093
0.143
IPI00154975
DNAJC9
DnaJ homolog subfamily C member 9
cyto
639
491
206
50
0.078
0.243
IPI00293260
DNAJC10
Isoform 1 of DnaJ homolog subfamily C member 10
nuc
834
51
687
48
0.058
0.070
IPI00465290
DNAJC11
Isoform 1 of DnaJ homolog subfamily C member 11
nuc
1490
182
649
164
0.110
0.253
IPI00307259
DNAJC13
DnaJ homolog subfamily C member 13
cyto
1585
613
138
289
0.182
2.090
IPI00006433
DNAJC16
Isoform 1 of DnaJ homolog subfamily C member 16
cyto
1539
1313
80
218
0.142
2.720
IPI00018798
DNAJC17
DnaJ homolog subfamily C member 17
nuc
284
21
238
0
Similar to DnaJ C17
nuc
19
0
107
1
0.027
0.005
IPI00936332
0.001
IPI00025510
DNAJC18
DnaJ homolog subfamily C member 18
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
Isoform 2 of DnaJ homolog subfamily C member 21
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
DnaJ homolog subfamily C member 30
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
Spatial Proteomics Sheds Light on the Biology of Nucleolar Chaperones
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Table 1. Contd…. Protein Accession
Gene
Description
Peptide Ion Intensity Localization
Relative abundance No/
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
Bag family molecular chaperone regulator 3
cyto
635
436
77
27
0.042
0.350
IPI00556027
BAG5
Isoform 2 of Bag family molecular chaperone regulator 5
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
T-complex protein 1 subunit delta
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
FK506-binding protein 10
nuc
3969
2166
3837
93
0.023
0.024
8 Current Proteomics, 2012, Vol. 9, No. 3
Kodiha et al.
Table 1. Contd…. Protein Accession
Gene
Description
Peptide Ion Intensity Localization
Relative abundance No/
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
Peptidyl-prolyl cis-trans isomerase-like 3
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
Protein disulfide-isomerase A5
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
Spatial Proteomics Sheds Light on the Biology of Nucleolar Chaperones
Table 2..
Current Proteomics, 2012, Vol. 9, No. 3
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
Hsp90 family IPI00382470
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
Heat shock protein 75 kD, mitochondrial
38.02
24.65
24.79
24.32
24.75
1.00
1.02
Hsp70 family IPI00643152
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
Heat shock 70 kD protein 1
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
Heat shock-related 70 kD protein 2
20.61
17.52
22.77
IPI00002966
HSPA4
Heat shock 70 kD protein 4
22.66
21.90
23.00
IPI00003362
HSPA5
HspA5 protein
20.64
19.52
20.13
23.69
1.15
1.18
IPI00339269
HSPA6
Heat shock 70 kD protein 6
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
Heat shock 70 kD protein 14
11.30
24.69
26.40
HYOU1
Hypoxia up-regulated protein 1
22.00
20.32
22.04
IPI00012535
DNAJA1
DnaJ homolog subfamily A member 1
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
DnaJ homolog subfamily B member 1
19.20
17.97
21.39
2.56
0.13
0.12
IPI00003848
DNAJB4
DnaJ homolog subfamily B member 4
7.62
15.57
18.72
DnaJs
10 Current Proteomics, 2012, Vol. 9, No. 3
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
DnaJ homolog subfamily B member 11
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
Isoform 1 of DnaJ C2
24.98
24.93
9.70
IPI00006713
DNAJC3
DnaJ homolog subfamily C member 3
18.34
21.61
17.45
IPI00402231
DNAJC5
Isoform 1 of DnaJ C5
18.20
17.26
18.72
IPI00329629
DNAJC7
DnaJ homolog subfamily C member 7
15.78
15.18
20.27
IPI00003438
DNAJC8
DnaJ homolog subfamily C member 8
18.17
17.04
18.97
18.76
1.03
0.99
IPI00154975
DNAJC9
DnaJ homolog subfamily C member 9
21.96
24.09
22.20
19.57
0.89
0.88
IPI00293260
DNAJC10
Isoform 1 of DnaJ C10
21.15
21.61
21.53
5.70
0.27
0.26
IPI00465290
DNAJC11
Isoform 1 of DnaJ C11
32.30
32.66
11.69
0.36
0.36
IPI00307259
DNAJC13
DnaJ homolog subfamily C member 13
23.08
21.81
22.61
3.87
0.17
0.17
IPI00006433
DNAJC16
Isoform 1 of DnaJ C16
20.15
12.92
1.15
IPI00018798
DNAJC17
DnaJ homolog subfamily C member 17
21.69
Similar to DnaJ C17
31.42
IPI00936332
15.57
0.09
22.45
IPI00025510
DNAJC18
DnaJ homolog subfamily C member 18
11.13
IPI00304306
DNAJC19
Mitochondrial import , translocase subunit TIM14
24.45
22.75
IPI00413366
DNAJC21
Isoform 2 of DnaJ C21
17.61
21.53
IPI00027909
DNAJC25
Isoform 1 of DnaJ C25
22.31
21.18
0.00
IPI00157375
DNAJC30
DnaJ homolog subfamily C member 30
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
Spatial Proteomics Sheds Light on the Biology of Nucleolar Chaperones
Current Proteomics, 2012, Vol. 9, No. 3
11
Table 2. Contd….
IPI00300531
Bag1
50% Turnover [h]
50% Turnover [h]
50% Turnover [h]
50% Turnover [h]
Relative turnover
Relative turnover
Isoform 1 of Bag family molecular chaperone regulator 1
18.79
21.12
12.84
20.20
1.08
1.57
IPI00000643
Bag2
Bag family molecular chaperone regulator 2
23.64
21.86
24.20
13.29
0.56
0.55
IPI00641582
Bag3
Bag family molecular chaperone regulator 3
15.43
15.09
14.96
15.41
1.00
1.03
IPI00556027
Bag5
Isoform 2 of Bag family molecular chaperone regulator 5
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
GrpE protein homolog 1, mitochondrial
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
Small heat shock proteins IPI00025512
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
Short heat shock protein 60 Hsp60s2
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
10 kD heat shock protein, mitochondrial
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
T-complex protein 1 subunit alpha
24.13
22.76
25.29
32.65
1.35
1.29
IPI00297779
CCT2
T-complex protein 1 subunit beta
24.92
22.69
26.62
30.10
1.21
1.13
IPI00553185
CCT3
T-complex protein 1 subunit gamma
22.77
22.10
25.04
12.08
0.53
0.48
IPI00302927
CCT4
T-complex protein 1 subunit delta
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
T-complex protein 1 subunit epsilon
17.27
14.99
21.69
11.64
0.67
0.54
IPI00027626
CCT6A
T-complex protein 1 subunit zeta
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
12 Current Proteomics, 2012, Vol. 9, No. 3
Kodiha et al.
Table 2. Contd…. 50% Turnover [h]
50% Turnover [h]
50% Turnover [h]
50% Turnover [h]
Relative turnover
Relative turnover
29.72
1.19
1.19
IPI00018465
CCT7
T-complex protein 1 subunit eta
25.05
22.72
25.02
IPI00784090
CCT8
T-complex protein 1 subunit theta
23.74
22.38
24.83
Additional factors involved in protein folding IPI00413778
FKBP1A
FKBP1A protein
24.53
24.19
3.69
IPI00219005
FKBP4
FK506-binding protein 4
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
FK506-binding protein 10
22.05
21.53
2.19
0.10
0.10
IPI00007019
PPIL1
Peptidyl-prolyl cis-trans isomerase-like 1
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
Protein disulfide-isomerase A3
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
Protein disulfide-isomerase A5
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
Ubiquitin carboxyl-terminal hydrolase 29
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
HECT domain containing 1
19.07
18.91
18.62
9.79
0.51
0.53
IPI00328911
HECTD1
E3 ubiquitin-protein ligase 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
Spatial Proteomics Sheds Light on the Biology of Nucleolar Chaperones
Current Proteomics, 2012, Vol. 9, No. 3
13
Table 2. Contd…. 50% Turnover [h]
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
Isoform 1 of 26S proteasome non-ATPase regulatory subunit 1
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.
14 Current Proteomics, 2012, Vol. 9, No. 3
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
Hsp70 family IPI00304925
HspA1A, HspA1B heat shock 70 kD protein 1
IPI00911039
Highly similar to heat shock 70 kD protein 1
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
Small heat shock proteins IPI00025512
HspB1 heat shock protein beta-1, Hsp27
Class I chaperonins IPI00784154
HspD1 60 kD heat shock protein, mitochondrial
Class II chaperonins IPI00290566
TCP1 T-complex protein 1 subunit alpha
IPI00302927,IPI00873222,IPI00893358,IPI0092 1414
CCT4 T-complex protein 1 subunit delta
Additional factors involved in protein folding IPI00025252
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
Spatial Proteomics Sheds Light on the Biology of Nucleolar Chaperones
Current Proteomics, 2012, Vol. 9, No. 3
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-
16 Current Proteomics, 2012, Vol. 9, No. 3
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
Putative heat shock protein Hsp90-beta 4
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
Heat shock protein Hsp90-beta
24.66
26.64
24.40
26.24
TRAP1
Heat shock protein 75 kD, mitochondrial
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
Heat shock 70 kD protein 4
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
Stress-70 protein, mitochondrial
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
Heat shock 70 kD protein 14
IPI00000877
HYOU1
Hypoxia up-regulated protein 1
IPI00012535
DNAJA1
DnaJ homolog subfamily A member 1
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
DnaJ homolog subfamily B member 1
17.93
19.74
39.89
17.97
21.39
2.56
0.06
IPI00830108
DNAJC2
Isoform 1 of DnaJ homolog subfamily C member 2
26.46
21.45
24.93
9.70
3.36
IPI00402231
DNAJC5
Isoform 1 of DnaJ homolog subfamily C member 5
16.95
21.85
17.26
18.72
IPI00465290
DNAJC11
Isoform 1 of DnaJ homolog subfamily C member 11
10.15
27.71
IPI00307259
DNAJC13
DnaJ homolog subfamily C member 13
IPI00304306
DNAJC19
Mitochondrial import inner membrane translocase subunit TIM14
22.13
IPI00413366
DNAJC21
Isoform 2 of DnaJ homolog subfamily C member 21
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
Spatial Proteomics Sheds Light on the Biology of Nucleolar Chaperones
Current Proteomics, 2012, Vol. 9, No. 3
19
Table 4. Contd….
Non-phosphorylated protein turnover
Phosphorylated protein turnover
[h]
[h]
phosphorylated/ nonphosphorylated
Small heat shock proteins IPI00025512
HSPB1
Heat shock protein beta-1, Hsp27
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
Class II chaperonins IPI00290566
TCP1
T-complex protein 1 subunit alpha
23.43
25.35
22.76
25.29
32.65
IPI00297779
CCT2
T-complex protein 1 subunit beta
22.38
26.46
29.79
22.69
26.62
30.10
1.01
IPI00553185
CCT3
T-complex protein 1 subunit gamma
21.79
24.25
22.28
22.10
25.04
12.08
0.54
IPI00302927
CCT4
T-complex protein 1 subunit delta
23.96
30.02
30.14
24.01
33.62
31.90
1.06
IPI00018465
CCT7
T-complex protein 1 subunit eta
22.94
24.72
47.40
22.72
25.02
29.72
0.63
IPI00784090
CCT8
T-complex protein 1 subunit theta
22.10
24.68
22.38
24.83
Additional factors involved in protein folding IPI00024157
FKBP3
FK506-binding protein 3
22.33
23.26
23.84
21.85
24.03
24.96
1.05
IPI00219005
FKBP4
FK506-binding protein 4
24.15
29.95
19.59
24.33
14.37
0.83
0.04
IPI00303300
FKBP10
FK506-binding protein 10
20.89
21.53
2.19
IPI00642862
PPIL4
Peptidyl-prolyl cis-trans isomerase-like 4
20.15
18.41
20.29
IPI00025252
PDIA3
Protein disulfide-isomerase A3
20.24
20.53
19.64
20.97
20.96
IPI00009904
PDIA4
Protein disulfide-isomerase A4
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
Ubiquitin-like modifier-activating enzyme 1
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
Ubiquitin carboxyl-terminal hydrolase 7
26.20
IPI00221012
USP9X
Ubiquitin specific protease 9, X-linked isoform 3
17.37
IPI00291946
USP10
Ubiquitin carboxyl-terminal hydrolase 10
22.61
12.33
22.27
26.37
0.94
0.86
1.10
1.63
19.91
3.91
20 Current Proteomics, 2012, Vol. 9, No. 3
Kodiha et al.
Table 4. Contd….
Non-phosphorylated protein turnover
Phosphorylated protein turnover
[h]
[h]
IPI00000728
USP15
Isoform 1 of Ubiquitin carboxyl-terminal hydrolase 15
18.66
22.82
IPI00045496
USP28
Isoform 1 of Ubiquitin carboxyl- terminal hydrolase 28
IPI00902614
USP24
Ubiquitin carboxyl-terminal hydrolase 24
IPI00001786
USP36
Isoform 2 of Ubiquitin carboxyl-terminal hydrolase 36
IPI00871372
HECTD1
HECT domain containing 1
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
Isoform 1 of 26S proteasome non-ATPase regulatory subunit 1
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
Spatial Proteomics Sheds Light on the Biology of Nucleolar Chaperones
Table 5.
Current Proteomics, 2012, Vol. 9, No. 3
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
Heat shock-related 70 kD protein 2
37.5
22.4
1.7
P38646
HSPA9
Stress-70 protein, mitochondrial
1.5
0.9
1.6
Q9Y4L1
HYOU1
Hypoxia up-regulated protein 1
6.5
3.3
2.0
DNAJA1
DnaJ homolog subfamily A member 1
3.2
3.6
0.9
Accession Hsp70 family
DnaJs P31689
Class II chaperonins P78371
CCT2
T-complex protein 1 subunit beta
2.9
2.7
1.1
P49368
CCT3
T-complex protein 1 subunit gamma
2.4
2.1
1.2
P48643
CCT5
T-complex protein 1 subunit epsilon
3.0
3.2
0.9
Protein disulfide-isomerase A4
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
E3 ubiquitin-protein ligase 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
22 Current Proteomics, 2012, Vol. 9, No. 3
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
Putative heat shock protein Hsp90-alpha A2
HSP90AB4P
Putative heat shock protein Hsp90-beta 4
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
Heat shock protein 75 kD, mitochondrial
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
Highly similar to heat shock 70 kD protein 1
0.22
0.12
0.37
0.39
0.03
0.13
0.34
HSPA4
Heat shock 70 kD protein 4
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
Hypoxia up-regulated protein 1
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
Spatial Proteomics Sheds Light on the Biology of Nucleolar Chaperones
Current Proteomics, 2012, Vol. 9, No. 3
23
Table 6. Contd….
Mock
No/Nuc
Etoposide
Eto/Mock
HSPBP1
Hsp70-binding protein 1
0.13
GrpEL1
GrpE protein homolog 1, mitochondrial
2.53
0.96
0.40
2.10
0.09
0.06
0.15
HSPB1
Heat shock protein beta-1, Hsp27
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
T-complex protein 1 subunit alpha
0.15
0.03
0.20
0.37
0.04
0.15
0.72
CCT2
T-complex protein 1 subunit beta
0.14
0.03
0.26
0.32
0.03
0.12
0.47
CCT3
T-complex protein 1 subunit gamma
0.16
0.03
0.16
0.34
0.04
0.16
0.95
CCT4
T-complex protein 1 subunit delta
0.16
0.04
0.21
0.37
0.03
0.11
0.54
CCT5
T-complex protein 1 subunit epsilon
0.15
0.04
0.19
0.34
0.06
0.21
1.10
CCT6A
T-complex protein 1 subunit zeta
0.17
0.03
0.18
0.44
0.05
0.17
0.96
CCT7
T-complex protein 1 subunit eta
0.15
0.04
0.25
0.36
0.05
0.16
0.64
CCT8
T-complex protein 1 subunit theta
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
E3 ubiquitin-protein ligase 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
24 Current Proteomics, 2012, Vol. 9, No. 3
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
Heat shock protein 75 kD, mitochondrial
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
GrpE protein homolog 1, mitochondrial
0.96
0.09
0.42
0.16
0.09
0.44
0.38
1.78
NP_005861
60 kD heat shock protein, mitochondrial
1.15
0.17
0.39
0.17
0.14
0.34
0.43
1.01
NP_065777
10 kD heat shock protein, mitochondrial
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
Heat shock protein 75 kD, mitochondrial
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
GrpE protein homolog 1, mitochondrial
0.70
Co-chaperones GRPEL1
2.02
Small heat shock proteins HSPB1
Heat shock protein beta-1, Hsp27
0.83
1.11
1.23
3.44
Class I chaperonins HSPD1
60 kD heat shock protein, mitochondrial
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
T-complex protein 1 subunit beta
1.07
1.70
CCT3
T-complex protein 1 subunit gamma
CCT4
T-complex protein 1 subunit delta
CCT5
T-complex protein 1 subunit epsilon
0.95
1.75
CCT6A
T-complex protein 1 subunit zeta
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
Spatial Proteomics Sheds Light on the Biology of Nucleolar Chaperones
Table 8.
Current Proteomics, 2012, Vol. 9, No. 3
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
Hsp90 family IPI00382470
Heat shock protein Hsp90-alpha
1.55
IPI00414676
Heat shock protein Hsp90-beta
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
DnaJ homolog subfamily C member 9
1.62
Hsp70 family
DnaJs
Class I chaperonins IPI00784154
Hsp60, 60 kD heat shock protein, mitochondrial precursor, CPN60
0.92
IPI00220362
Hsp10, 10 kD heat shock protein, mitochondrial, CPN10
0.49
IPI00290566
T-complex protein 1 subunit alpha
0.96
IPI00297779
T-complex protein 1 subunit beta
1.12
IPI00553185
T-complex protein 1 subunit gamma
1.12
IPI00873222
T-complex protein 1 subunit delta;
1.14
IPI00027626
T-complex protein 1 subunit zeta, CCT6A
1.11
Class II chaperonins
Additional factors involved in protein folding IPI00149650
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.
26 Current Proteomics, 2012, Vol. 9, No. 3
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
Putative heat shock protein Hsp90-alpha A2
HSP90AB1
Heat shock protein beta (Fragment)
HSP90AB1
Heat shock protein Hsp90-beta (Hsp90, Hsp84)
HSP90B1
Endoplasmin, Grp94
Hsp70 family HSPA4
Heat shock 70 kD protein 4
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
Hypoxia up-regulated protein 1
DnaJs DNAJA1, HDJ2
DnaJ homolog subfamily A member 1
DNAJA2
DnaJ homolog subfamily A member 2
DNAJB1
DnaJ homolog subfamily B member 1 (Hsp40)
DNAJC9
DnaJ homolog subfamily C member 9
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
Short heat shock protein 60 Hsp60s2
HSPD1 (HSP60)
60 kD heat shock protein, mitochondrial, Hsp60, CPN60
HSPE1
10 kD heat shock protein, mitochondrial, Hsp10, CPN10
Class II chaperonins TCP1, CCT1
T-complex protein 1 subunit alpha
CCT2
T-complex protein 1 subunit beta
CCT3
T-complex protein 1 subunit gamma
CCT4
T-complex protein 1 subunit delta
CCT5
T-complex protein 1 subunit epsilon
CCT6A
T-complex protein 1 subunit zeta
CCT7
T-complex protein 1 subunit eta
CCT8
T-complex protein 1 subunit theta
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)
Spatial Proteomics Sheds Light on the Biology of Nucleolar Chaperones
Current Proteomics, 2012, Vol. 9, No. 3
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)
Ubiquitin-like modifier-activating enzyme 1
UHRF1
E3 ubiquitin-protein ligase UHRF1 (EC 6.3.2.-)
USP7 (HAUSP)
Ubiquitin carboxyl-terminal hydrolase 7 (EC 3.1.2.15)
USP11
Ubiquitin carboxyl-terminal hydrolase 11 (EC 3.1.2.15)
USP14
Ubiquitin carboxyl-terminal hydrolase 14 (EC 3.1.2.15)
USP21
Ubiquitin carboxyl-terminal hydrolase 21 (EC 3.1.2.15)
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
28 Current Proteomics, 2012, Vol. 9, No. 3
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
Spatial Proteomics Sheds Light on the Biology of Nucleolar Chaperones
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
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Accepted: 00 00, 2012