The human TPR protein TTC4 is a putative Hsp90 co-chaperone which interacts with CDC6 and shows alterations in transformed cells

Abstract

Background: The human TTC4 protein is a TPR (tetratricopeptide repeat) motif-containing protein. The gene was originally identified as being localized in a genomic region linked to breast cancer and subsequent studies on melanoma cell lines revealed point mutations in the TTC4 protein that may be associated with the progression of malignant melanoma.

Methodology/principle findings: Here we show that TTC4 is a nucleoplasmic protein which interacts with HSP90 and HSP70, and also with the replication protein CDC6. It has significant structural and functional similarities with a previously characterised Drosophila protein Dpit47. We show that TTC4 protein levels are raised in malignant melanoma cell lines compared to melanocytes. We also see increased TTC4 expression in a variety of tumour lines derived from other tissues. In addition we show that TTC4 proteins bearing some of the mutations previously identified from patient samples lose their interaction with the CDC6 protein.

Conclusions/significance: Based on these results and our previous work with the Drosophila Dpit47 protein we suggest that TTC4 is an HSP90 co-chaperone protein which forms a link between HSP90 chaperone activity and DNA replication. We further suggest that the loss of the interaction with CDC6 or with additional client proteins could provide one route through which TTC4 could influence malignant development of cells.

Figure 1. a. Comparison of the protein sequence of humanTTC4 (Hs) with its Drosophila (Dm) and S. Cerevisiae (Sc) orthologues.

Areas of identity are shown in shading. The region of the protein containing the TPR domains is underlined. Point mutations of TTC4 which were tested for their interaction with hCDC6 and Hsp90 and 70 (Fig 6) are coloured in red a-42 (V to D), b-67(I to M),c-77 (E to A), and d-217(N to K). b. Phylogenetic tree showing a comparison between the TTC4 protein and a variety of other TPR containing proteins which interact with Hsp90 and/or Hsp70. Each protein is tagged with a prefix to show which species the protein is from h-human, Dm–Drosophila melanogaster, Sc–S. cerevisiae.

Figure 2. TTC4 shows interaction with human Hsp90 and 70 using the yeast 2-hybrid system.

Pairs of bait and activation vectors as indicated were co-transfected into S. cerevisiae and plated as described in Materials and Methods. The growth of each on selective and non-selective media after 1 day is shown.

Figure 3. TTC4 shows interaction with human HSP90, HSP70 and CDC6 by immunoprecipitation from Hela cells extract.

a: TTC4 serum recognises a single band of 48 kDa in whole cell extracts (lane 1). The intensity of this band is greatly reduced upon competition with GST TTC4 (lane 2). Molecular weight standards are indicated on the left in kDa. b: HSP90, HSP70 and CDC6 co immunoprecipitate with TTC4. The immunoprecipitation was carried out as indicated in Materials and Methods. WCE: Whole cell extract, snt pre immune: supernatant after incubation with the pre immune serum bound to protein A sepharose, snt IP: supernatant after incubation with the anti TTC4 immune serum bound to protein A sepharose, pellet pre immune: eluate from the pre immune serum bound to protein A sepharose, pellet IP eluate from the anti TTC4 immune serum bound to protein A sepharose. For WCE, snt pre immune and snt IP: 40 µg were loaded on the gel. For pellet, pre immune and pellet IP: 1/5 of each eluate was loaded on the gel.

Figure 4. Mutations affecting the TPR domain of TTC4 disrupt binding to Hsp90.

The wild type and mutant TTC4 proteins were mixed with HSP90 and incubated as described in Methods. The left panel of this Coomassie stained PAGE shows the purified proteins used in the assay (Inputs) and the right hand panel the glutathione beads resuspended in SDS PAGE loading buffer after co incubation of the proteins (pellets). Wt: wild type GST-TTC4, K: K152E GST-TTC4 mutant, R1 and R2: R156E GST-TTC4 mutant and RK: K152E, R156E GST-TTC4 mutant. More sample was loaded into the lanes with mutant proteins in an effort to detect faint interactions.

Figure 5. TTC4 shows interaction with human cdc6 using the yeast 2-hybrid system.

Pairs of bait and activation vectors as indicated were co-transfected into S. cerevisiae and plated as described in materials and methods. The growth of each on selective and non-selective media after 2 days is shown.

Figure 6. TTC4 is predominantly a nuclear protein.

a. Indirect immunofluorescence staining of Hermes 3a and Nohm1 cells was carried out as described in Materials and Methods. In each case TTC4 is stained in red, tubulin in green and DNA in blue. b. Detail of the individual staining patterns of tubulin and TTC4 in Nohm1 cells. c. Immunoblot to show the location of TTC4 after fractionation of Hela cells into cytoplasmic (cyt), nucleoplasm (np) and chromatin associated (pellet) fractions as described in Materials and Methods. Alpha-tubulin (immunoblot) and Histones (Coomassie- blue staining) are shown as controls for the fractionation. Note that the coomassie stained band in the cytoplasmic lane of the histone panel is not histone related.

Figure 7. TTC4 expression is higher in rapidly dividing tissue and is also altered in tumour derived cell lines.

a. Immunoblot showing the expression of TTC4 in human placental (P1 and P2) and heart (Ht) samples. b and c. Immunoblots showing the expression of TTC4 in a number of tumour derived cell lines. N: normal (MRC5 human lung fibroblast, A: A2780 (ovarian adenocarcinoma), M:MDA (breast), L:Lovo (colon carcinoma), H: HT29 (colon adenocarcinoma), P;PC3 (prostate adenocarcinoma), J: Jurkat (acute t cell leukaemia), Mc: MCF7 ( breast carcinoma) He:Hela (cervical carcinoma). Tubulin loading controls are shown for both blots. d. Immunoblot showing a comparison of the expression of TTC4 in melanocytes (No: Nohm 1 and Hr :Hermes 3a), and malignant melanoma lines (Dx:DX3 and Lt: LT5.1). e. Immunblot comparing the relative levels of TTC4 in placenta and transformed cells (Hela). N: normal (MRC5 human lung fibroblast), H:hela and P1 placenta.