The Cytosolic Chaperonin CCT/TRiC and Cancer Cell Proliferation

<div><p>The molecular chaperone CCT/TRiC plays a central role in maintaining cellular proteostasis as it mediates the folding of the major cytoskeletal proteins tubulins and actins. CCT/TRiC is also involved in the oncoprotein cyclin E, the Von Hippel-Lindau tumour suppressor protein, cyclin B and p21^ras folding which strongly suggests that it is involved in cell proliferation and tumor genesis. To assess the involvement of CCT/TRiC in tumor genesis, we quantified its expression levels and activity in 18 cancer, one non-cancer human cell lines and a non-cancer human liver. We show that the expression levels of CCT/TRiC in cancer cell lines are higher than that in normal cells. However, CCT/TRiC activity does not always correlate with its expression levels. We therefore documented the expression levels of CCT/TRiC modulators and partners PhLP3, Hop/P60, prefoldin and Hsc/Hsp70. Our analysis reveals a functional interplay between molecular chaperones that might account for a precise modulation of CCT/TRiC activity in cell proliferation through changes in the cellular levels of prefoldin and/or Hsc/p70 and CCT/TRiC client protein availability. Our observation and approaches bring novel insights in the role of CCT/TRiC-mediated protein folding machinery in cancer cell development.</p></div

CCT/TriC, Hop/p60 or PhLP3 immunodepletion affects actin refolding.

<p>CCT/TRiC folding activity in (<b>A</b>) MIA-PaCa-2 and (<b>C</b>) OVCAR-8<b> </b>cell extracts before and after CCT/TriC, Hop/p60 or PhLP3 immunodepletion was assayed using [³⁵S]-labeled, denatured β-actin. The protein concentration of the MIA-PaCa-2 and OVCAR-8 cell extracts was 2.5 and 15 mg/ml, respectively. Native β-actin band intensity was measured using a PhosphorImager. The amount of native β-actin measured after immunodepletion is expressed as a fraction of the amount measured for the cell extracts before immunodepletion in (<b>B</b>) MIA-PaCa-2 and (<b>D</b>) OVCAR-8 cell extracts.</p

CCT/TriC activity modulators in cancer cell lines extracts.

<p>The amount of (<b>A</b>) Hop/p60, (<b>B</b>) PhLP3 and (<b>C</b>) PFD in 18 cancer and a normal (MRC5-SV2) cell lines extracts, a non-cancer liver homogenate (HNCL) and rabbit reticulocyte lysate (RRL) was determined by comparing the chemiluminescence signal recorded for Hop/p60, PhLP3 and PFD in cell extracts diluted 4 and 16 fold to that of known amounts of the proteins on the same Western blots.</p

Characteristics of the cell lines and cell extracts used throughout this study.

<p>Characteristics of the cell lines and cell extracts used throughout this study.</p

Hsc/p 70 expression in cancer cell lines extracts.

<p>The amount of Hsc/p70 in 18 cancer and a normal (MRC5-SV2) cell lines extracts, a liver homogenate (HNCL) and rabbit reticulocyte lysate (RRL) was determined by comparing the chemiluminescence signal recorded for Hsc/p70 in cell extracts diluted 4 and 16 fold to that of known amounts of the protein on the same Western blots. The average amounts of Hsc/p70 in the different extracts are expressed as a function of the total proteins content of the extracts.</p

Hsp/c70 immunodepletion affects CCT/TRiC-mediated β-actin folding.

<p>CCT/TRiC-mediated labeled, denatured β-actin folding in (<b>A</b>) MIA-PaCa-2 and OVCAR-8 cell extracts before and after Hsc/p70 immunodepletion. The protein concentration of the MIA-PaCa-2 and OVCAR-8 cell extracts was 2.5 and 15 mg/ml, respectively. Native β-actin band intensity was recoded using a PhosphorImager. The amount of native β-actin measured after immunodepletion is expressed as a fraction of the amount measured for the cell extracts before immunodepletion in (<b>B</b>) MIA-PaCa-2 and (<b>C</b>) OVCAR-8 cell extracts.</p

Soluble Ure2p is a proteasomal substrate <i>in vitro</i>.

<p><b>(A)</b> Purified 26S proteasomes (2 nM) were mixed with purified soluble Ure2p (250 nM) (upper panel) or Sup35p (125 nM) (lower panel) in the presence of 2.5 mM ATP, with or without MG132 (100 μM), as indicated. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). At the indicated time points, aliquots were removed and analyzed by SDS-PAGE and Western blotting using anti-Ure2p or anti-Sup35p antibodies. Ure2p* and Sup35p* indicate proteasome-resistant fragments. <b>(B)</b> Purified 26S proteasomes (2 nM) were mixed with purified Ure2p (250 nM) and without or with increasing concentrations of purified Sup35p (250 nM to 1 μM), in the presence of 2.5 mM ATP. Reaction mixes were incubated and analyzed as described in (A). <b>(C)</b> Purified 26S proteasomes (2 nM) were mixed with purified Sup35p (125 nM) and without or with increasing concentrations of purified Ure2p (250 nM to 500 nM), in the presence of 2.5 mM ATP. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). Reaction mixes were incubated and analyzed as described in (A).</p

Ure2p degradation by the 26S proteasome yields ~10–30 aminoacids-long peptides originating from the N-terminal end of the protein.

<p>Purified soluble Ure2p (1 μg) was incubated without or with purified 26S proteasome (0.4 μg) in the presence of 2.5 mM ATP at 30°C under mild agitation (<300 rpm) for 0, 1, 2 or 3 h. Peptides produced during the incubation were identified by nanoLC-LTQ-Orbitrap mass spectrometry (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131789#pone.0131789.s001" target="_blank">S1 Table</a>). These peptides were not produced when Ure2p was incubated alone (data not shown). The color code reflects the time point at which each individual peptide was first detected, as indicated. <b>(Inset)</b> Aliquots from the reaction mixes were analyzed by SDS-PAGE and Western blot using anti-Ure2p antibodies.</p

The deletion of residues 3–25 prevents the proteasomal degradation of Ure2p.

<p><b>(A)</b> Cartoon representation of the Ure2p variants used in this study. <b>(B)</b> Time-courses of Ure2p, Ure2Δ3–25 and Ure2Δ1–93 (25 μM) assembly at 6°C, monitored by thioflavin T binding (a.u., arbitrary units). Data represent the mean of three independent experiments ± SE <b>(C)</b> Negative-stained electron micrographs of Ure2p, Ure2Δ3–25 and Ure2Δ1–93 (25 μM) assemblies after 30 days of incubation at 6°C (scale bar: 500 nm). Only amorphous aggregates were detected for Ure2Δ1–93. <b>(D)</b> Purified Ure2p, Ure2Δ3–25 or Ure2Δ1–93 (250 nM) were incubated with or without purified 26S proteasomes (2 nM), as indicated, and in the presence of 2.5 mM ATP. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). At the indicated time points, aliquots were removed from the reaction mix and analyzed by SDS-PAGE and Western blotting using anti- Ure2p antibodies.</p

Schematic of a polyQ.

<p>A polyQ contains a minimum of five consecutive Q residues. The maximum proportion of residues other than Q (insertions) is 25% and each insertion cannot be over 5-residues long. The N- and C-terminal flanks of the polyQ are labeled Nt and Ct flanks, respectively. The numbering scheme for the residues within the flanks is shown.</p