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. 2020 Sep 4;26(1):265–274. doi: 10.1007/s12192-020-01156-3

Analysis of insect nuclear small heat shock proteins and interacting proteins

Mohamed Taha Moutaoufik 1,2, Robert M Tanguay 1,✉
PMCID: PMC7736433  PMID: 32888179

Abstract

The small heat shock proteins (sHsps) are a ubiquitous family of ATP-independent stress proteins found in all domains of life. Drosophila melanogaster Hsp27 (DmHsp27) is the only known nuclear sHsp in insect. Here analyzing sequences from HMMER, we identified 56 additional insect sHsps with conserved arginine-rich nuclear localization signal (NLS) in the N-terminal region. At this time, the exact role of nuclear sHsps remains unknown. DmHsp27 protein-protein interaction analysis from iRefIndex database suggests that this protein, in addition to a putative role of molecular chaperone, is likely involved in other nuclear processes (i.e., chromatin remodeling and transcription). Identification of DmHsp27 interactors should provide key insights on the cellular and molecular functions of this nuclear chaperone.

Electronic supplementary material

The online version of this article (10.1007/s12192-020-01156-3) contains supplementary material, which is available to authorized users.

Keywords: Small heat shock protein (sHsp), DmHsp27, Chaperone, Alpha-crystallin domain (ACD), Drosophila melanogaster, Insect

Introduction

The nucleus is the cellular organelle that distinguishes eukaryotes from prokaryotes. A large number of biological activities occur in the nucleus, including DNA replication and damage repair, the biogenesis of ribosomal RNA precursors, transcription, and splicing of pre-mRNAs. Many proteins are involved in nuclear transport in processes dependent on Karyopherin-β proteins (both importins and exportins).

The small heat shock proteins (sHsps) are a ubiquitous family of ATP-independent stress proteins found in all domains of life (Caspers et al. 1995; de Jong et al. 1998; Fu et al. 2006; Maaroufi and Tanguay 2013). These proteins are upregulated in response to a variety of stresses that negatively impact protein homeostasis. sHsp sequence analysis indicates a tripartite architecture, with a conserved α-crystallin domain (ACD) flanked by a variable non-conserved N- and C-terminal regions (NTR and CTR) (Kappé et al. 2010; Basha et al. 2012). sHsp number, level and stage of expression, tissue distribution, and intracellular localization can show differences between species, suggesting that sHsps have adapted to their environment and to specific cell needs (Michaud et al. 2002; Morrow and Tanguay 2015).

Relocation of cytosolic sHsps to the nucleus is observed under certain conditions of stress in virtually all mammals (Van De Klundert and De Jong 1999; Borrelli et al. 2002), but this is distinct from nuclear sHsps that are present all the time in the nucleus in Drosophila melanogaster and plant, due to the presence of a nuclear localization signal (NLS) (Beaulieu et al. 1989; Marin and Tanguay 1996; Scharf et al. 2001; Siddique et al. 2003, 2008; Michaud et al. 2008).

Determining the cellular localization of proteins is important for understanding protein functions. Here we focused on the nuclear-localized small Hsp27 of Drosophila melanogaster (DmHsp27). This sHsp was the first reported to be localized in the nucleus (Beaulieu et al. 1989), and since this discovery, other sHsps, especially from plants, have been shown to have a nuclear localization such as Solanum peruvianum LpHsp16.1, Arabidopsis thaliana AtHsp17.4, Gossypium arboreum GaHsp17.3, Medicago truncatula MtHsp17.1, Lactuca sativa LsHsp17.8, Triticum aestivum TaHsp19.0, Hordeum vulgare HvHsp19.1, and Oryza sativa OsHsp18.6 (Scharf et al. 2001; Siddique et al. 2003, 2008). The import of proteins into the nucleus is dependent on a nuclear localization signals (NLS), and members of the nuclear transport receptor (importin β-like) superfamily. In plants, the nuclear localization depends on a short motif (N(G)KRKR) located between β-strands 5 and 6 in the conserved ACD (Siddique et al. 2003). This mode requires importin α to serve as a bridge between the NLS and the import receptor importin β (Marfori et al. 2012). In Drosophila melanogaster DmHsp27, the NLS motif is composed of three arginine (Arg54-Arg55-Arg56) in the NTR (Michaud et al. 2008). Authors suggested that arginine 54 and 55 form a single functional group that acts in concert with arginine 56 to dictate nuclear localization of DmHsp27 (Michaud et al. 2008). Arginine-rich NLS uses importin β for import by a mechanism independent of importin α (Palmeri and Malim 1999). The difference between the nuclear localization of sHsps in mammals, plants, and D. melanogaster suggests that they adapt to their environments and their specific cellular needs. While we know the existence of several sHsps with an NLS in plants, DmHsp27 is the only member reported in insect. NTR in sHsp was reported as a disordered non-conserved region due to the lack of sequence conservation and structure among this family (Kim et al. 1998; van Montfort et al. 2001). Therefore, localization of a nuclear signal in this disordered and non-conserved region is surprising. Here we characterize nuclear insect sHsps containing an arginine-rich NLS and suggest possible functions of this group of sHsps based on DmHsp27 interacting partners.

Materials and methods

Sequence collection of the insect sHsps

All insect protein sequences containing one or more instances of ACD (accession number PF00011) were extracted from HMMER web server (Finn et al. 2011). Proteins containing the term “fragment” in their description were removed. From this data, we extracted only sequences containing arginine-rich NLS (residues XRR and RXR) in NTR as defined in Drosophila melanogaster by Michaud et al. (2008). After alignment using ClustalW (Larkin et al. 2007), only proteins showing a conservation of NLS with DmHsp27 have been kept.

Lengths, amino acid composition, alignment, and logos of insect sHsp

The beginning and end locations ACD of all sequences were extracted from PFAM database (Finn et al. 2014). The sequences were split into the 3 building blocks (ACD, NTR, and CTR). The length frequencies were analyzed using R ggplot2 package (Wickham 2009), and amino acid distribution average was evaluated using R stringi package (https://cran.r-project.org/web/packages/stringi/).

The sequences were aligned with ClustalW default parameters. Secondary structure of the alignment generated by ESPript (Robert and Gouet 2014) according to DmHsp27 ACD predicted structure by Moutaoufik et al. (2017b). UniProt accession numbers of sequences used in the alignments are indicated. To identify conserved regions, we used WebLogo 3 program (Crooks et al. 2004) to create blocks (LOGOS) of conserved amino acid residues from the multiple protein sequences.

Phylogenetic analysis of the insect sHsps

Multiple alignments of insect sHsp sequences were calculated in MEGAX ((Kumar et al. 2018)) using ClustalW default parameters. Maximum likelihood phylogenetic trees were constructed using MEGAX default parameters and 50 bootstrap replicates. Phylogenetic trees were visualized using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/).

Protein-protein interaction data

In order to understand nuclear functions of insect sHsps, we searched protein-protein interaction (PPI) data of insect nuclear sHsps using iRefIndex database (Razick et al. 2008). From all other insect species identified in this study, only Drosophila melanogaster PPI data was available in iRefIndex. DmHsp27 experimentally verified PPI data were downloaded, filtered by nuclear localization, and imported into Cytoscape (Shannon et al. 2003) to construct PPI network.

Results and discussion

Nucleus plays a central role in eukaryotes. However, its protein content is far from complete. A mouse nuclear proteomic prediction reveals that mouse nucleus is the house of between 4084 and 9122 proteins (Fink et al. 2008). Using FlyBase (Thurmond et al. 2019) and UniProt Gene Ontology (GO) (2019), we identified reported nuclear proteins in Drosophila melanogaster. The total number of nuclear proteins reported in FlyBase and UniProt Go is constant with 2348 and 2344 entries, respectively (Fig. 1a, Supplementary Table 1), although more than half proteins (1479) are shared between two databases.

Fig. 1.

Fig. 1

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Drosophila melanogaster proteome and insect sHsps. A Venn diagram showing common and specific Drosophila melanogaster nuclear proteins between public FlyBase and UniProt GO databases. B Frequency of nuclear sHsp length. Full length sequence presented in (gray), NTR (blue), ACD (red), and CTR (green). C Nuclear sHsp evolutionary analysis by Maximum Likelihood method. Nuclear sHsps highlighted in red

Collection of nuclear insect sHsp sequences

It is accepted that the presence of the conserved ACD is a sufficient criterion for assigning a sequence to the sHsp family (Caspers et al. 1995; Kappé et al. 2010). To collect the sequences of sHsps, we searched the presence of ACD by using the domain PF00011 in HMMER web server (Finn et al. 2011). We obtained 1321 insect sequences containing ACD (Supplementary Table 2), with an average length of 188.82 (± 26.89) (Fig. 1b). NTR have a length average of 74.50 (± 20.67) and CTR 23.98 (± 16.26); both show a variation in the length and frequency (Fig. 1b). ACD less variable region with a length average of ACD 90.33 (± 7.95).

Analysis of NTR shows that 57 sequences contain an arginine-rich NLS in NTR (Table 1). The potential nuclear localization was confirmed by Euk-mPLoc (Chou and Shen 2007), a powerful tool, developed by hybridizing the functional domain information and sequential evolutionary information through three different modes of pseudo amino acid composition (Eq.4, Eq.6, and Eq.12).

Table 1.

Insect nuclear sHsps

Protein name UniProt ID Species Length Mass (Dalton) Arginine-rich NLS localization
Hsp 27 P02518 Drosophila melanogaster 213 23,617 Arg 54,55,56
GM25143 B4HKP4 Drosophila sechellia 213 23,625 Arg 54,55,56
GD14177 A0A0J9UHX1 Drosophila simulans 213 23,625 Arg 54,55,56
GD14177 B4QN53 Drosophila simulans 213 23,566 Arg 54,55,56
GG15371 B3NCF2 Drosophila erecta 212 23,650 Arg 54,55,56
GE20833 B4PEX9 Drosophila yakuba 212 23,594 Arg 54,55,56
ND A0A1I8M7V4 Musca domestica 216 24,395 Arg 52–53
Hsp20 T1PAE8 Musca domestica 216 24,364 Arg 52–53
Hsp27 A0A1L8EIU6 Haematobia irritans 203 23,200 Arg 48–50
Hsp27 A0A1L8EIY3 Haematobia irritans 203 23,250 Arg 48–50
Hsp27 A0A1L8EJ25 Haematobia irritans 203 23,327 Arg 48–50
Hsp27 A0A1L8EJ27 Haematobia irritans 203 23,223 Arg 48–50
Hsp27 A0A1L8EJ69 Haematobia irritans 203 23,350 Arg 48–50
Hsp27 A0A1L8EJ80 Haematobia irritans 203 23,212 Arg 48–50
Hsp27 A0A1X9JRV5 Musca domestica 203 23,031 Arg 47–49
ND A0A1I8M631 Musca domestica 203 23,040 Arg 47–49
Hsp27 A0A1L8EJ49 Haematobia irritans 203 23,261 Arg 48–50
Hsp27 A0A1L8EIP8 Haematobia irritans 202 23,112 Arg 49–50–51
Hsp27 A0A1L8EIV9 Haematobia irritans 202 23,126 Arg 49–50–51
Hsp27 A0A1L8EJ16 Haematobia irritans 202 23,098 Arg 49–50–51
Hsp27 A0A1L8EIS9 Haematobia irritans 203 22,956 Arg 48–50
Hsp27 A0A1L8EIV6 Haematobia irritans 203 23,117 Arg 48–50
Hsp27 A0A1L8EIW9 Haematobia irritans 203 23,078 Arg 48–50
Hsp27 A0A1L8EIX8 Haematobia irritans 203 23,097 Arg 48–50
Hsp27 A0A1L8EIY9 Haematobia irritans 203 23,022 Arg 48–50
Hsp27 A0A1L8EJ10 Haematobia irritans 203 23,068 Arg 48–50
Hsp27 A0A1L8EJ18 Haematobia irritans 203 23,038 Arg 48–50
Hsp27 A0A1L8EJ35 Haematobia irritans 203 23,123 Arg 48–50
Hsp27 A0A1L8EJ40 Haematobia irritans 203 23,105 Arg 48–50
Hsp27 A0A1L8EJ47 Haematobia irritans 203 23,127 Arg 48–50
ND T1PK02 Musca domestica 198 22,646 Arg 47–49
Hsp27 A0A1X9JUH8 Musca domestica 198 22,628 Arg 47–49
Hsp27 A0A0L0CSL4 Lucilia cuprina 203 23,331 Arg 47–49–50
GK17637 B4MN48 Drosophila willistoni 227 25,285 Arg 58,59,60
Hsp27 A9UEZ2 Drosophila buzzatii 211 23,695 Arg 51,52,53
ND A0A1B0G3A3 Glossina morsitans morsitans 212 24,164 Arg 55–57
ND A0A1A9UGE3 Glossina austeni 184 21,061 Arg 55–57
Hsp27 I1T1H1 Bactrocera dorsalis 210 23,831 Arg 51–52–53
Hsp27 A0A0K8UP97 Bactrocera latifrons 212 24,037 Arg 53–54–-55
Hsp27 A0A0A1XCZ4 Bactrocera cucurbitae 212 23,920 Arg 53–54–55
Hsp27 B3GK93 Ceratitis capitata 214 23,944 Arg 54–55–56
Hsp27 A0A0M4EGE5 Drosophila busckii 217 24,445 Arg 51–52–53
Hsp27 A0A3B0JUV4 Drosophila guanche 225 25,016 Arg 58–59–60
GL22445 B4H1L8 Drosophila persimilis 225 25,041 Arg 58,59,60
GA18205 Q29F98 Drosophila pseudoobscura pseudoobscura 225 25,041 Arg 58,59,60
GF10692 B3M6F4 Drosophila ananassae 219 24,490 Arg 57,58,59
Hsp27 F5B960 Drosophila albomicans 222 24,770 Arg 52,53,54
Hsp27 F5B961 Drosophila sulfurigaster albostrigata 221 24,675 Arg 52,53,54
GJ13835 B4LGS9 Drosophila virilis 211 23,792 Arg 51,52,53
GH17009 B4IYY1 Drosophila grimshawi 218 24,442 Arg 54,55,56
Hsp25 A1E385 Sarcophaga crassipalpis 221 24,982 Arg 60–61
ND A0A484BBT9 Drosophila navojoa 211 23,628 Arg 51–52–53
GI13087 B4L112 Drosophila mojavensis 209 23,459 Arg 51,52,53
Hsp27 F5B962 Drosophila repletoides 220 24,504 Arg 48,49,50
CSON012237 A0A336MGY1 Culicoides sonorensis 167 19,445 Arg 40–41
CSON11881 A0A336MMP2 Culicoides sonorensis 167 19,401 Arg 40–41
Hsp27 A0A1W4VN65 Drosophila ficusphila 213 23,784 Arg 53–54–55
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Next, we studied the evolutionary history of insect sHsp sequences (Fig. 1c). It is interesting that insect nuclear sHsps were clustered in one phytogenic group, suggesting that all nuclear insect sHsps evolved from the same ancestral sequence.

Analysis of insect nuclear sHsps

Insect sHsp sequences that presented arginine-rich NLS include drosophila genus: Drosophila simulans (2 sequences) and 1 sequence for Drosophila albomicans, Drosophila ananassae, Drosophila busckii, Drosophila buzzatii, Drosophila erecta, Drosophila ficusphila, Drosophila grimshawi, Drosophila guanche, Drosophila melanogaster, Drosophila mojavensis, Drosophila navojoa, Drosophila persimilis, Drosophila pseudoobscura pseudoobscura, Drosophila repletoides, Drosophila sechellia, Drosophila sulfurigaster albostrigata, Drosophila virilis, Drosophila willistoni, and Drosophila yakuba. In addition to other genera: Haematobia irritans (20 sequences), Musca domestica (6 sequences), Culicoides sonorensis (2 sequences) and 1 sequence for Bactrocera cucurbitae, Bactrocera dorsalis, Bactrocera latifrons, Ceratitis capitate, Glossina austeni, Glossina morsitans morsitans, Lucilia cuprina, and Sarcophaga crassipalpis (Table 1) suggesting that arginine-rich NLS is not specific to drosophila.

The average mass in Dalton is 23,489.00 (± 1086.55). The average amino acid length is 207.77 (± 11.45). The longest sequence is B4MN48 from Drosophila willistoni with 227 amino acids (aa) and the shortest is A0A336MGY1 from Culicoides sonorensis with sequence 167 aa (Table 1).

The average length of amino acids in NTR is 84.40 (± 2.61) (Fig. 2a). The analysis of NTR revealed underrepresentation of cysteine and an overrepresentation of leucine, proline, aspartic acid, and arginine (Fig. 2a and b). ACD has an average length of 88.56 (± 2.74) (Fig. 2a). In contrast, within the ACD, a significant enrichment of charged positively (lysine) and negatively (aspartic acid and glutamic acid) residues is observed, whereas tryptophan is absent (Fig. 2a and c). Most sequences present one cysteine in ACD, suggesting that under oxidative conditions, a disulfide crosslink may be formed between ACDs to form a dimer as shown for DmHsp27 (Moutaoufik et al. 2017b). In CTR, the shortest region has an average amino acid number of 34.80 (± 1.50) with absence of tryptophan, phenylalanine, and tyrosine. Additionally, there is underrepresentation of cysteines and arginine (Fig. 2a and d). This is consistent with a previous study from 1935 metazoan proteins which contain ACD by Kriehuber et al. (2010).

Fig. 2.

Fig. 2

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Insect nuclear sHsps. A Distribution of residues within the nuclear sHsps in insect. Average number of each amino acid within the NTR (blue), CTR (green), and the ACD (red) is presented. B, C, and D Logo presentation using WebLogo 3 program (Crooks et al. 2004). The height of each letter is made proportional to its frequency. Amino acids are colored according to their chemical properties: acidic (D,E) in red, basic (K,R,H) in blue, polar (G,S,T,Y,C) in green and (N,Q) in purple, and hydrophobic (A,V,L,I,P,W,F,M) in black. Logo representation for Logos presentation of NTR (B), ACD (C), and CTR (D) of insect nuclear sHsp sequences

Blocks of the most conserved residues were made as logos to visualize the conserved regions in three regions—NTR, ACD, CTR—of nuclear insect sHsps (Fig. 2c, d, and e) in addition to a complete sequence alignment which (Supplementary Fig. 1) shows that motif characteristics of sHsps were conserved. This is the case of the arginine in the Beta 6 + 7 strand: DmHsp27-R122 equivalent of HspB1-R127, DmHsp27-R131 equivalent of HspB1-R136, and DmHsp27-R135 equivalent of HspB1-R140, HspB5-R120, and HspB4-R116 associated with human pathologies (Vicart et al. 1998; Bera and Abraham 2002; Evgrafov et al. 2004; Inagaki et al. 2006; Gentil and Cooper 2012; Ylikallio et al. 2015). In addition to the conserved I/V/L-X-I/V/L motif in ACD, implicated in sHsps higher assembly by interaction with β4/β8 pocket in ACD of adjacent dimer (de Jong et al. 1998; van Montfort et al. 2001; Stamler et al. 2005; Poulain et al. 2010; Clark et al. 2018) and the FGFG motif in NTR, important for the oligomeric structure and chaperone-like activity (Moutaoufik et al. 2017a). In addition, B4PEX9 (Drosophila yakuba), B4HKP4 (Drosophila sechellia), B3NCF2 (Drosophila erecta), A1E385 (Sarcophaga crassipalpis), and A0A0J9UHX1 and B4QN53 (Drosophila simulans) show a conservation in both phosphorylated serines in DmHsp27 (S58 and S75) reported by Moutaoufik et al. (2017a). T1PAE8 (Musca domestica), B4L112 (Drosophila mojavensis), A9UEZ2 (Drosophila buzzatii), A0A1I8M7V4 (Musca domestica), and A0A484BBT9 (Drosophila navojoa) show only a conservation in phosphorylated serine (S58). However, A0A1L8EIU6, A0A1L8EIY3, A0A1L8EJ25, A0A1L8EJ27, A0A1L8EJ69, A0A1L8EJ80, A0A1L8EJ49, A0A1L8EIP8, A0A1L8EIV9, A0A1L8EJ16, A0A1L8EIS9, A0A1L8EIV6, A0A1L8EIW9, A0A1L8EIX8, A0A1L8EIY9, A0A1L8EJ10, A0A1L8EJ18, A0A1L8EJ35, A0A1L8EJ40 and A0A1L8EJ47 (Haematobia irritans), A0A1X9JRV5, A0A1I8M631, T1PK02 and A0A1X9JUH8 (Musca domestica), A0A3B0JUV4 (Drosophila guanche), B3M6F4 (Drosophila ananassae), A0A1W4VN65 (Drosophila ficusphila), A0A0L0CSL4 (Lucilia cuprina), A0A1B0G3A3 (Glossina morsitans morsitans), A0A1A9UGE3 (Glossina austeni), Q29F98 (Drosophila pseudoobscura pseudoobscura), B4MN48 (Drosophila willistoni), and B4H1L8 (Drosophila persimilis) show only a conservation in phosphorylated serine (S75).

DmHsp27 interactome and nuclear functions

The nuclear functions of insect sHsps remain unknown. A study had reported an association of DmHsp22, DmHsp23, DmHsp26, and DmHsp27 to heterogeneous nuclear ribonucleoprotein (hnRNP) (Kloetzel and Bautz 1983). In addition, under heat shock conditions, DmHsp27 and cytoplasmic DmsHsps were co-purified with the proteasome (Arrigo et al. 1985).

We searched the protein partners of insect sHsps identified in this study, using iRefIndex (Razick et al. 2008). Only Drosophila melanogaster protein-protein interactions are presented in the used database. DmHsp27 shows 46 associations with both nuclear and cytoplasmic proteins. After filtering by subcellular localization, we identified 24 nuclear proteins that tend to interact with DmHsp27 in addition to self-interacting to form oligomeric structures (Fig. 3, Table 2). Third (8, 33%) of DmHsp27 nuclear partners were involved in regulation of transcription, more than one-quarter (7, 29%) in ATPase activity, (2, 0.08%) were implicated in chromatin remodeling, (1, 0.04%) in ubiquitin proteasome complex and quarter (6, 25%) with no known nuclear function (Fig. 3b). These include proteins involved in many biological processes such as fly development (14-3-3ε, Lk6, enhancer of zeste E(Z), centrosomin (cnn), lissencephaly-1 (Lis-1), lesswright (lwr), p53, slow border cells (slbo), and suppressor of variegation 3–9 (Su(var)3–9)) (Ashton-Beaucage et al. 2014; Arquier et al. 2005; Jones et al. 1998; Eisman et al. 2009; Liu et al. 1999; Miles et al. 2008; Bauer et al. 2007; Borghese et al. 2006; Kunert et al. 2003). In addition, LIM homeobox 1 (Lim1) implicated in eye development (Roignant et al. 2010). However, 14-3-3ε, Alhambra (Alh), and transcriptional adaptor 2 Ada2b were associated with locomotor rhythm and muscle and wing development (Bahri et al. 2001; Gause et al. 2006; Bejarano et al. 2008). Finally, 14-3-3ε, p53, and CG5436 (Hsp68) were related to fly viability (Bauer et al. 2005; Nielsen et al. 2008; Biteau et al. 2010), lwr to immune system response (Bhaskar et al. 2002), and CG5436 (Hsp68), Hsp70Aa, and Hsp70Ba to chaperone activity (Gong and Golic 2006; Gaudet et al. 2011). This is consistent with the related expression of DmHsp27 during oogenesis and spermatogenesis (Marin and Tanguay 1996; Michaud et al. 1997), eye development (Chen et al. 2012), lifespan (Hao et al. 2007; Liao et al. 2008), and defense against pathogens (Chen et al. 2010).

Fig. 3.

Fig. 3

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DmHsp27 and its nuclear-associated proteins. Representation of DmHsp27 and associated protein assemblies. Green color indicates transcription regulators, purple color designate proteins with ATPase activity, blue color indicate proteins with non-defined nuclear functions, red color indicate chromatin remodeler, and gray indicates protein involved in ubiquitin proteasome system

Table 2.

Proteins associated with DmHsp27

Protein 1 UniprotID1 Protein 2 UniprotID2 Method Reference
14-3-3ε P92177 DmHsp27 P02518 Colocalization Müller et al. (2010)
Ada2b Q8I8V0 Dmhsp27 P02518 Two-hybrid Giot et al. (2003)
Alh Q9VI61 DmHsp27 P02518 Two-hybrid Giot et al. (2003)
CG18004 A1Z8D4 DmHsp27 P02518 AP-MS Guruharsha et al. (2011)
CG3726 X2JDV1 DmHsp27 P02518 AP-MS Rhee et al. (2014)
CG8950 Q7JVW5 DmHsp27 P02518 AP-MS Rhee et al. (2014)
CG9596 Q8MS69 DmHsp27 P02518 AP-MS Guruharsha et al. (2011)
CG5436 O97125 DmHsp27 P02518 AP-MS Guruharsha et al. (2011)
CNN P54623 DmHsp27 P02518 MS Habermann et al. (2012)
E(z) P42124 DmHsp27 P02518 AP-MS Kang et al. (2015)
DmHsp27 P02518 DmHsp27 P02518 BN gel Moutaoufik et al. (2017a)
Hsp70 Aa P82910 Dmhsp27 P02518 AP-MS Guruharsha et al. (2011)
Hsp70Ba Q8INI8 DmHsp27 P02518 AP-MS Guruharsha et al. (2011)
Lim1 M9PJE4 DmHsp27 P02518 Two-hybrid Giot et al. (2003)
Lis1 Q7KNS3 DmHsp27 P02518 Two-hybrid Giot et al. (2003)
Lk6 Q9VGI4 DmHsp27 P02518 Two-hybrid Giot et al. (2003)
Lwr Q7KNM2 DmHsp27 P02518 AP-WB/Two-hybrid Joanisse et al. (1998)
p53 A0A0B4K7P1 DmHsp27 P02518 AP-WB Lei et al. (2017)
Rpt1 Q7KMQ0 DmHsp27 P02518 AP-MS Guruharsha et al. (2011)
Ref(2)P P14199 DmHsp27 P02518 AP-MS Guruharsha et al. (2011)
RpS27A P15357 DmHsp27 P02518 AP-WB Lee et al. (2014)
Rpt5 Q9V3V6 DmHsp27 P02518 AP-WB Cho-Park and Steller (2013)
Slbo Q02637 DmHsp27 P02518 Genetic Interactions Rørth et al. (1998)
Su(var)3–9 I0DHL3 DmHsp27 P02518 Two-hybrid Giot et al. (2003)
Xport-A Q9VDS3 DmHsp27 P02518 AP-WB Rosenbaum et al. (2011)
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Conclusion

Here we have shown that the NLS signal rich in arginine is not a peculiarity of Drosophila melanogaster. The progress of genome sequencing projects should confirm the presence of other insect sHsp sequences with NLS signal, of the same type or not. The exact role of nuclear sHsps remains unknown. The interaction network of DmHsp27 suggests that this protein does not only play the role of molecular chaperone, but it is likely involved in different nuclear processes. Such studies open the perspectives to establish the functional activities of DmHsp27 and associated proteins in different biological processes.

Electronic supplementary material

Supplementary Figure 1 (2.8MB, png)

Sequence alignment of nuclear drosophila small heat shock proteins. Alignment was generated using ClustalW. UniProt accession numbers of sequences used in the alignments are indicated. Secondary structures of the alignment generated by ESPript (Robert and Gouet 2014), according to DmHsp27 ACD predicted structure by Moutaoufik et al. 2017b. NTRs show conservation of FGFG motif and arginine-rich NLS (Black arrow). The I/V/L-X-I/L/V motif in the CTR and conserved arginine R122, R131, and R135 are indicated by black arrow and black star, respectively. (PNG 2876 kb).

High resolution image (TIF 12054 kb). (11.8MB, tif)
Supplementary Table 1 (839.5KB, xls)

(XLS 839 kb).

Supplementary Table 2 (212.5KB, xls)

(XLS 212 kb).

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure 1 (2.8MB, png)

Sequence alignment of nuclear drosophila small heat shock proteins. Alignment was generated using ClustalW. UniProt accession numbers of sequences used in the alignments are indicated. Secondary structures of the alignment generated by ESPript (Robert and Gouet 2014), according to DmHsp27 ACD predicted structure by Moutaoufik et al. 2017b. NTRs show conservation of FGFG motif and arginine-rich NLS (Black arrow). The I/V/L-X-I/L/V motif in the CTR and conserved arginine R122, R131, and R135 are indicated by black arrow and black star, respectively. (PNG 2876 kb).

High resolution image (TIF 12054 kb). (11.8MB, tif)
Supplementary Table 1 (839.5KB, xls)

(XLS 839 kb).

Supplementary Table 2 (212.5KB, xls)

(XLS 212 kb).

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